Biofilm prevention, disruption and treatment with bacteriophage lysin

ABSTRACT

The present invention provides methods for the prevention, control, disruption and treatment of bacterial biofilms with lysin, particularly lysin having capability to kill Staphylococcal bacteria, including drug resistant  Staphylococcus aureus , particularly the lysin PlySs2. The invention also provides compositions and methods for use in treatment or modulation of bacterial biofilm(s) and biofilm formation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/391,272 (allowed), filed Apr. 22, 2019, which is a continuation ofU.S. patent application Ser. No. 15/293,586, filed on Oct. 14, 2016,which is a Continuation of application Ser. No. 14/399,588, filed Nov.7, 2014 (now U.S. Pat. No. 9,499,594), which was filed as PCTInternational Application No. PCT/US2013/040340 on May 9, 2013, whichclaims the benefit under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 61/736,813, filed on Dec. 13, 2012, and U.S. ProvisionalApplication No. 61/644,799, filed on May 9, 2012, all of which arehereby expressly incorporated by reference into the present application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on 11 Nov. 2022, isnamed 0341_0008.03.xml and is 7 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates generally to prevention, control,disruption and treatment of bacterial biofilms with lysin, particularlylysin having capability to kill Staphylococcal bacteria, including drugresistant Staphylococcus aureus, particularly the lysin PlySs2. Theinvention also relates to compositions and methods for modulation ofbacterial biofilm(s) and biofilm formation.

BACKGROUND OF THE INVENTION

The development of drug resistant bacteria is a major problem inmedicine as more antibiotics are used for a wide variety of illnessesand other conditions. The use of more antibiotics and the number ofbacteria showing resistance has prompted longer treatment times.Furthermore, broad, non-specific antibiotics, some of which havedetrimental effects on the patient, are now being used more frequently.A related problem with this increased use is that many antibiotics donot penetrate mucus linings easily.

Gram-positive bacteria are surrounded by a cell wall containingpolypeptides and polysaccharide. Gram-positive bacteria include but arenot limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus,Staphylococcus, Streptococcus, Enterococcus, Mycobacterium,Corynebacterium, and Clostridium. Medically relevant species includeStreptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus,and Enterococcus faecalis. Bacillus species, which are spore-forming,cause anthrax and gastroenteritis. Spore-forming Clostridium species areresponsible for botulism, tetanus, gas gangrene and pseudomembranouscolitis. Corynebacterium species cause diphtheria, and Listeria speciescause meningitis.

Novel antimicrobial therapy approaches include enzyme-based antibiotics(“enzybiotics”) such as bacteriophage lysins. Phages use these lysins todigest the cell wall of their bacterial hosts, releasing viral progenythrough hypotonic lysis. A similar outcome results when purified,recombinant lysins are added externally to Gram-positive bacteria. Thehigh lethal activity of lysins against gram-positive pathogens makesthem attractive candidates for development as therapeutics (Fischetti,V. A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al(2001) Proc Natl Acad Sci USA 98:4107-4112). Bacteriophage lysins wereinitially proposed for eradicating the nasopharyngeal carriage ofpathogenic streptococci (Loeffler, J. M. et al (2001) Science 294:2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112).Lysins are part of the lytic mechanism used by double stranded DNA(dsDNA) phage to coordinate host lysis with completion of viral assembly(Wang, I. N. et al (2000) Annu Rev Microbiol 54:799-825). Lysins arepeptidoglycan hydrolases that break bonds in the bacterial wall, rapidlyhydrolyzing covalent bonds essential for peptidoglycan integrity,causing bacterial lysis and concomitant progeny phage release.

Lysin family members exhibit a modular design in which a catalyticdomain is fused to a specificity or binding domain (Lopez, R. et al(1997) Microb Drug Resist 3:199-211). Lysins can be cloned from viralprophage sequences within bacterial genomes and used for treatment(Beres, S. B. et al (2007) PLoS ONE 2(8):1-14). When added externally,lysins are able to access the bonds of a Gram-positive cell wall(Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400).Bacteriophage lytic enzymes have been established as useful in theassessment and specific treatment of various types of infection insubjects through various routes of administration. For example, U.S.Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection ofGroup A streptococci in clinical specimens, through the enzymaticdigestion by a semi-purified Group C streptococcal phage associatedlysin enzyme. This enzyme work became the basis of additional research,leading to methods of treating diseases. Fischetti and Loomis patents(U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use ofa lysin enzyme produced by group C streptococcal bacteria infected witha C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis)discloses a composition for dermatological infections by the use of alytic enzyme in a carrier suitable for topical application to dermaltissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses amethod for treatment of bacterial infections of the digestive tractwhich comprises administering a lytic enzyme specific for the infectingbacteria. The carrier for delivering at least one lytic enzyme to thedigestive tract is selected from the group consisting of suppositoryenemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945(Fischetti and Loomis) discloses a method and composition for thetreatment of bacterial infections by the parenteral introduction(intramuscularly, subcutaneously, or intravenously) of at least onelytic enzyme produced by a bacteria infected with a bacteriophagespecific for that bacteria and an appropriate carrier for delivering thelytic enzyme into a patient.

Phage associated lytic enzymes have been identified and cloned fromvarious bacteriophages, each shown to be effective in killing specificbacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCTApplication WO2008/018854 provides distinct phage-associated lyticenzymes useful as antibacterial agents for treatment or reduction ofBacillus anthracis infections. U.S. Pat. No. 7,569,223 describes lyticenzymes for Streptococcus pneumoniae. Lysin useful for Enterococcus (E.faecalis and E. faecium, including vancomycin resistant strains) aredescribed in U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutantPly GBS lysins highly effective in killing Group B streptococci. Achimeric lysin denoted ClyS, with activity against Staphylococcibacteria, including Staphylococcus aureus, is detailed in WO2010/002959. ClyS is specific for Staphylococcal bacteria and isinactive against Streptococcus and other gram positive bacteria.

Based on their rapid, potent, and specific cell wall-degradation andbactericidal properties, lysins have been suggested as antimicrobialtherapeutics to combat Gram-positive pathogens by attacking the exposedpeptidoglycan cell walls from outside the cell (Fenton, M et al (2010)Bioengineered Bugs 1:9-16; Nelson, D et al (2001) Proc Natl Acad Sci USA98:4107-4112). Efficacies of various lysins as a single agents have beendemonstrated in rodent models of pharyngitis (Nelson, D et al (2001)Proc Natl Acad Sci USA 98:4107-4112), pneumonia (Witzenrath, M et al(2009) Crit Care Med 37:642-649), otitis media (McCullers, J. A. et al(2007) PLOS pathogens 3:0001-0003), abscesses (Pastagia, M et alAntimicrobial agents and chemotherapy 55:738-744) bacteremia (Loeffler,J. M. et al (2003) Infection and Immunity 71:6199-6204), endocarditis(Entenza, J. M. et al (2005) Antimicrobial agents and chemotherapy49:4789-4792), and meningitis (Grandgirard, D et al (2008) J Infect Dis197:1519-1522). In addition, lysins are generally specific for theirbacterial host species and do not lyse non-target organisms, includinghuman commensal bacteria which may be beneficial to gastrointestinalhomeostasis (Blaser, M. (2011) Nature 476:393-394; Willing, B. P. et al(2011) Nature reviews. Microbiology 9:233-243)

Microorganisms tend to form surface-attached biofilm communities as animportant survival strategy in different environments. Biofilms consistof microbial cells and a wide range of self-generated extracellularpolymeric substances, including polysaccharides, nucleic acids, andproteins (Flemming H C et al (2007) J Bacteriol 189:7945-7947). Biofilmsare found in natural and industrial aquatic environments, tissues, andmedical materials and devices (Costerton J W et al (1994) J Bacteriol176:2137-2142). Biofilms can be formed by a single bacterial strain,although most natural biofilms are formed by multiple bacterial species(Yang L et al (2011) Int J Oral Sci 3:74-81). Applications ofantibiotics are often ineffective for biofilm populations due to theirunique physiology and physical matrix barrier.

Staphylococci often form biofilms, sessile communities encased in anextracellular matrix that adhere to biomedical implants or damaged andhealthy tissue. Infections associated with biofilms are difficult totreat, and it is estimated that sessile bacteria in biofilms are 1,000to 1,500 times more resistant to antibiotics than their planktoniccounterparts. This antibiotic resistance of biofilms often leads to thefailure of conventional antibiotic therapy and necessitates the removalof infected devices. Lysostaphin has been shown to kill S. aureus inbiofilms and also disrupted the extracellular matrix of S. aureusbiofilms in vitro on plastic and glass surfaces (Wu, J A et al (2003)Antimicrob Agents and Chemoth 47(11):3407-3414). This disruption of S.aureus biofilms was specific for lysostaphin-sensitive S. aureus, andbiofilms of lysostaphin-resistant S. aureus were not affected. Highconcentrations of oxacillin (400 μg/ml), vancomycin (800 μg/ml), andclindamycin (800 μg/ml) had no effect on the established S. aureusbiofilms, even after 24 h. Lysostaphin also disrupted S. epidermidisbiofilms, however, higher concentrations were required. Application ofphage lysins for the removal of staphylococcal biofilms have beenreported, with mixed results. Bacteriophage lysin SAL-2 was reported toremove S. aureus biofilms (Son J S et al (2010) Appl MicrobiolBiotechnol 86(5):1439-1449), while in the case of two similar phagelysins, phi11 and phi12, while phi11 hydrolyzed staphylococcal biofilms,phi12 was inactive (Sass P and Bierbaum G (2007) Appl Environ Microbiol73(1):347-352). Various combinations of enzymes have been studied forthe removal and disinfection of bacterial biofilms in various systems(Johansen C et al (1997) Appl Environ Microbiol 63:3724-3728). Thisprocess, however, requires a minimum of two enzymes or agents, oneenzyme or agent for removal of the adherent bacteria of the biofilms anda second enzyme or agent with bactericidal activity.

It is evident from the deficiencies and problems associated with currenttraditional antibacterial agents that there still exists a need in theart for additional specific bacterial agents and therapeutic modalitiesand also for broader spectrum agents, particularly without risks ofacquired resistance, for the effective and efficient treatment, controland prevention of bacterial biofilms. It is notable that to date, nolysin demonstrating lytic activity against multiple distinct species ofpathogenic and clinically relevant gram positive bacteria, which isreadily manufacturable and stable, and has no or limited risk ofresistance, has been shown to be effective on biofilms. Accordingly,there is a commercial need for new antibacterial approaches, especiallythose that operate via new modalities or provide new means to killpathogenic bacteria in biofilms.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided for the prevention, disruption and treatment of bacterialbiofilms. In its broadest aspect, the present invention provides use andapplication of a lysin having broad killing activity against multiplebacteria, particularly Gram-positive bacteria, including particularlyStaphylococcus, Streptococcus, particularly Streptococcus pyogenes(Group A strep) and Streptococcus agalactiae (Group B strep) bacterialstrains, in the prevention, disruption and treatment of biofilms. Thelysin and compositions of the invention are useful and applicable inkilling Enterococcus and Listeria bacterial strains, and in applicablebiofilms thereof. The invention provides a method for decolonizing,dispersing and removal of bacterial biofilm utilizing bacteriophagelysin capable of killing bacteria effectively and efficiently in abiofilm. The invention thus contemplates treatment, decolonization,and/or decontamination of bacterial biofilms and the prevention ofinfections after dispersion of biofilm(s) wherein one or more grampositive bacteria, particularly one or more of Staphylococcus,Streptococcus, Enterococcus and Listeria bacteria, is suspected orpresent.

In accordance with the present invention, bacteriophage lysin derivedfrom Streptococcus suis bacteria are utilized in the methods andapplications of the invention. The lysin polypeptide(s) of use in thepresent invention, particularly PlySs2 lysin as provided herein and inFIG. 5 (SEQ ID NO: 1), are unique in demonstrating broad killingactivity against multiple bacteria, particularly gram-positive bacteria,including Staphylococcus, Streptococcus, Enterococcus and Listeriabacterial strains. In one such aspect, the PlySs2 lysin is capable ofkilling Staphylococcus aureus strains and bacteria in biofilms, asdemonstrated herein. PlySs2 is effective against antibiotic-resistantbacteria, including Staphylococcus aureus such as methicillin-resistantStaphylococcus aureus (MRSA), vancomycin resistant Staphylococcus aureus(VRSA), daptomycin-resistant Staphylococcus aureus (DRSA) andlinezolid-resistant Staphylococcus aureus (LRSA). PlySs2 is effectiveagainst bacteria with altered antibiotic sensitivity such as vancomycinintermediate-sensitivity Staphylococcus aureus (VISA).

In an aspect of the invention, a method is provided of killinggram-positive bacteria in a biofilm comprising the step of contactingthe biofilm with a composition comprising an amount of an isolated lysinpolypeptide effective to kill gram-positive bacteria in a biofilm,including S. aureus, the isolated lysin polypeptide comprising thePlySs2 lysin polypeptide or variants thereof effective to killgram-positive bacteria. Thus, a method is provided of killinggram-positive bacteria in a biofilm comprising the step of contactingthe biofilm with a composition comprising an amount of an isolated lysinpolypeptide effective to kill the gram-positive bacteria in the biofilm,the isolated lysin polypeptide comprising the amino acid sequenceprovided in FIG. 5 or SEQ ID NO: 1 or variants thereof having at least80% identity, 85% identity, 90% identity, 95% identity or 99% identityto the polypeptide of FIG. 5 or SEQ ID NO: 1 and effective to kill thegram-positive bacteria in the biofilm.

In an aspect of the invention, a method is provided of dispersinggram-positive bacteria in a biofilm so as to decontaminate and torelease bacteria then susceptible to antibiotics, comprising the step ofcontacting the biofilm with a composition comprising an amount of anisolated lysin polypeptide effective to disperse gram-positive bacteriain a biofilm, including S. aureus, the isolated lysin polypeptidecomprising the PlySs2 lysin polypeptide, including as set out in FIG. 5or SEQ ID NO: 1 or variants thereof effective to kill gram-positivebacteria.

In an aspect of the above methods, the methods are performed in vitro orex vivo so as to sterilize or decontaminate a solution, material ordevice, particularly intended for use by or in a human.

The invention provides a method for reducing a population ofgram-positive bacteria in a biofilm comprising the step of contactingthe biofilm with a composition comprising an amount of an isolatedpolypeptide effective to kill or release at least a portion of thegram-positive bacteria in the biofilm, the isolated polypeptidecomprising the amino acid sequence of FIG. 5 (SEQ ID NO: 1) or variantsthereof having at least 80% identity to the polypeptide of FIG. 5 (SEQID NO: 1) and effective to kill the gram-positive bacteria.

The present invention further provides a method for dispersing ortreating an antibiotic-resistant Staphylococcus aureus infection whichinvolves or includes a biofilm in a human comprising the step ofadministering to a human with an antibiotic-resistant Staphylococcusaureus biofilm infection, an effective amount of a compositioncomprising an isolated polypeptide comprising the amino acid sequence ofFIG. 5 (SEQ ID NO: 1) or variants thereof having at least 80% identity,85% identity, 90% identity or 95% identity to the polypeptide of FIG. 5(SEQ ID NO: 1) and effective to disperse the biofilm and killStaphylococcus aureus therein and//or released therefrom, whereby thenumber of Staphylococcus aureus in the human is reduced and the biofilmand attendant infection is controlled.

A method of the invention also includes a method for preventing,dispersing or treating a gram-positive bacterial biofilm comprising oneor more of Staphylococcus or Streptococcus bacteria in a humancomprising the step of administering to a subject having or suspected ofhaving or at risk of a bacterial biofilm, an effective amount of acomposition comprising an isolated polypeptide comprising the amino acidsequence of FIG. 5 (SEQ ID NO: 1) or variants thereof having at least80% identity, 85% identity, 90% identity or 95% identity to thepolypeptide of FIG. 5 (SEQ ID NO: 1) and effective to kill thegram-positive bacteria, whereby the number of gram-positive bacteria inthe human is reduced and the biofilm contamination or infection iscontrolled. In an aspect of the method, biofilm comprising or includingone or more of an Enterococcus or Listeria bacteria is effectivelyprevented, dispersed or treated. In a particular aspect of this method,wherein the subject is exposed to or at risk of one of or one or more ofStaphylococcus (such as Staphylococcus aureus), Streptococcus(particularly Group A strep or Group B strep such as Streptococcuspyogenes or Streptococcus agalactiae, respectively) bacteria. Analternative bacteria such as Listeria (such as L. monocytogenes) orEnterococcus (such as E. faecalis) bacteria may also be involved andaddressed, prevented, dispersed or treated in accordance with themethods and compositions of the invention. The subject may be a human.The subject may be a human adult, child, infant or fetus.

In any such above method or methods, the susceptible, killed, dispersedor treated biofilm bacteria may be selected from Staphylococcus aureus,Listeria monocytogenes, Staphylococcus simulans, Streptococcus suis,Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo,Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcusdysgalactiae, Group G Streptococcus, Group E Streptococcus, Enterococcusfaecalis and Streptococcus pneumonia.

In accordance with any of the methods of the invention, the susceptiblebacteria or biofilm bacteria may be an antibiotic resistant bacteria.The bacteria may be antibiotic resistant, includingmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistantStaphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus(DRSA), or linezolid-resistant Staphylococcus aureus (LRSA). Thebacteria may have altered antibiotic sensitivity, such as for example,vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), Thesusceptible bacteria may be a clinically relevant or pathogenicbacteria, particularly for humans. In an aspect of the method(s), thelysin polypeptide(s) is effective to kill Staphylococcus, Streptococcus,Enterococcus and Listeria bacterial strains.

It has been shown that coating medical implants with antimicrobials mayeffectively prevent the initial adherence of staphylococcal biofilms tothe implants. Coating biomedical materials with lysin may also provesuccessful in preventing early adherence of bacteria, includingstaphylococci, to the implants, thus averting biofilm formation. Thepresent invention thus also provides methods for reducing or preventingbiofilm growth on the surface of devices, implants, separation membranes(for example, pervaporation, dialysis, reverse osmosis, ultrafiltration,and microfiltration membranes) by administering or coating with thelysin of the invention, including PlySs2 lysin.

Alternative active and suitable lysin(s) may be utilized in accordancewith the methods and compositions of the present invention, including asthe lysin(s) of use and/or as one or more additional effective anduseful lysins. In an additional aspect or embodiment of the methods anduses provided herein, the staphylococcal specific lysin ClyS is usedherein alone or in combination with the PlySs2 lysin as provided anddescribed herein.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts biofilms of BAA-42 MRSA treated with daptomycin,vancomycin, PlySs2 lysin or linezolid at the amounts and for the timesindicated up to 4 hours. Antibiotics daptomycin, vancomycin, andlinezolid were added at 1000×MIC for each antibiotic. PlySs2 was addedat 1×MIC. After treatment, biofilms are visualized with crystal violet.

FIG. 2 depicts biofilms of BAA-42 MRSA treated with daptomycin,vancomycin, PlySs2 lysin or linezolid at the amounts and for the timesindicated up to 6 hours. After treatment, biofilms are visualized withcrystal violet.

FIG. 3 depicts biofilms of BAA-42 MRSA treated with daptomycin,vancomycin, PlySs2 lysin or linezolid at the amounts and for the timesindicated up to 24 hours. After treatment, biofilms are visualized withcrystal violet.

FIG. 4 depicts biofilms of BAA-42 MRSA in 24 well dishes treated withPlySs2 lysin or daptomycin for 0.5 hr, 1 hr, 4 hrs and 24 hrs at theindicated dosing amounts. After treatment, biofilms are visualized withcrystal violet.

FIG. 5 provides the amino acid sequence (SEQ ID NO: 1) and encodingnucleic acid sequence (SEQ ID NO: 2) of the lysin PlySs2. The N-terminalCHAP domain and the C-terminal SH-3 domain of the PlySs2 lysin areshaded, with the CHAP domain starting with LNN . . . and ending with . .. YIT (SEQ ID NO: 3) and the SH-3 domain starting with RSY . . . andending with . . . VAT (SEQ ID NO: 4). The CHAP domain active-siteresidues (Cys₂₆, His₁₀₂, Glu₁₁₈, and Asn₁₂₀) identified by homology toPDB 2K3A (Rossi P et al (2009) Proteins 74:515-519) are underlined.

FIG. 6 provides a twenty-four hour time course analysis of PlySs2 andantibiotic activity on MRSA biofilms as assessed by crystal violetstaining. Antibiotics daptomycin (DAP), vancomycin (VAN) and linezolid(LZD) were added at 1000×MIC for each antibiotic. PlySs2 was added at1×MIC.

FIG. 7 depicts quantitation of dye retained as an indicator of biofilmretained in a twenty-four hour time course analysis of PlySs2 andantibiotic activity on MRSA biofilms. Antibiotics daptomycin (DAP),vancomycin (VAN) and linezolid (LZD) were added at 1000×MIC for eachantibiotic. PlySs2 lysin was added at 1×MIC.

FIG. 8 shows a 24 hour time course of sub-MIC concentrations of PlySs2versus media alone on MRSA biofilms as assessed by crystal violetstaining. PlySs2 was added to MRSA strain BAA-42 biofilm at 0.1×MIC and0.01×MIC levels.

FIGS. 9A and 9B depict biofilm eradication studies against MRSA grown onDEPC catheters. FIG. 9A: Catheter biofilms were treated with mediaalone, 1×MIC daptomycin, 1000×MIC daptomycin and 1×MIC PlySs2 for 24hours before flushing, staining with methylene blue and photographing.FIG. 9B: After 24 hours of treatment, duplicate catheter samples weretreated with lysis buffer to remove residual biofilms and bacterial CFUsestimated based on relative light units using a luciferase reagentcalibrated against known concentrations of bacteria.

FIG. 10 depicts titration analysis of DEPC catheter MRSA biofilmstaining with methylene blue after 4 hour treatment with buffer ortitrated MICs of PlySs2 of 1×MIC, 0.1×MIC, 0.01×MIC, 0.001×MIC,0.0001×MIC and 0.00001×MIC PlySs2.

FIG. 11 depicts titration analysis of DEPC catheter MRSA biofilmstaining with methylene blue after 4 hour treatment with buffer ortitrated daptomycin (DAP) at 5000×MIC, 1000×MIC, 100×MIC, 10×MIC and1×MIC.

FIGS. 12A and 12B show a time course analysis of PlySs2 activity againstMRSA biofilms in DEPC catheters. FIG. 12A: catheters were treated with1×MIC PlySs2 (32 ug/ml) for 5 min, 15 min, 30 min, 60 min, 90 min, 2hrs, 3 hrs, 4 hrs and 5 hrs before flushing, staining with methyleneblue and photographing. FIG. 12B: After each timed treatment, duplicatecatheter samples were treated with lysis buffer to remove residualbiofilms and bacterial CFUs estimated based on relative light unitsusing a luciferase reagent calibrated against known concentrations ofbacteria.

FIG. 13 depicts a titration analysis of DEPC catheter MRSA biofilm CFUcounts after 4 hour treatments of catheter biofilms with the indicateddrug concentrations in accordance with the studies shown in FIGS. 11 and12 . Bacterial CFUs remaining after drug treatments were estimated basedon relative light units using a luciferase reagent calibrated againstknown concentrations of bacteria. Biofilms formed by Staphylococcusaureus strain ATCC BAA-42 on the lumens of di(2-ethylhexyl)phthalate(DEHP) catheters were treated for 4 hours with the indicatedconcentrations of PlySs2 or daptomycin (DAP). Lactated Ringer's solutionalone was included as a control. After treatment, the catheters weredrained and washed, and colony-forming units (CFU) were measured usingan adenosine triphosphate (ATP) release-based method (BacTiter-Glo™Microbial Cell Viability Assay kit). The red line indicates theconcentrations of DAP at 5000× the minimum inhibitory concentration(MIC) and PlySs2 at 0.01×MIC that resulted In roughly equivalentdecreases in biofilms in the treated catheter tubes. Key: *=Below thethreshold of detection.

FIG. 14 depicts lysin ClyS activity against S. aureus biofilm. Biofilmsof BAA-42 MRSA were treated with the indicated concentrations of ClySlysin (1×MIC 32 μg/ml, 0.1×MIC 3.2 μg/ml, 0.01×MIC 0.32 μg/ml and0.001×MIC 0.032 μg/ml) or media alone for 24 hours. Each well was washedand stained with 2% crystal violet.

FIG. 15 provides the results of biofilm studies in vivo in mice withsubcutaneous catheter implants treated with PlySs2 lysin by variousmodes of administration. Biofilms are grown on catheters, the catheteris implanted in mice, and the mice are treated. Catheters are removed,stained with methylene blue and staining quantified by absorbance at 600nm. The OD at 600 nm/g of catheter is graphed for each of negativecontrol (no bacteria), PlySs2 control (no bacteria mock treated),vehicle treated, PlySs2 administered intraperitoneally (IP), PlySs2administered intravenously (IV), and PlySs2 administered subcutaneously(SC).

FIG. 16 depicts time course studies evaluating the luminal contents ofMRSA catheter biofilms treated with PlySS2 lysin or daptomycin andassessing for bacterial viability and luminal sterilization over timewith treatment of PlySs2 or antibiotic daptomycin.

FIG. 17 depicts titration analysis of a catheter study withStaphylococcal epidermidis strain CFS 313 (NRS34, a VISE strain)bacterial biofilm. Biofilm staining with methylene blue is shown after 4hour treatment with buffer or titrated MICs of PlySs2 of 10×MIC, 1×MIC(8 μg/ml), 0.1×MIC, 0.01×MIC, 0.001×MIC and 0.0001×MIC PlySs2.

FIG. 18 depicts a biofilm prevention assay of BAA-42 MRSA bacteriainoculated in 24 well plates and combined immediately with buffer orPlySs2 at 1×MIC (32 μg/ml), or dilutions noted to 0.0001×MIC. The plateswere incubated for 6 hours, washed with PBS, stained with crystal violetto evaluate biofilm generation and photographed.

FIG. 19 depicts titration analysis of catheter MRSA strain CFS 553 (ATCC43300) biofilm staining with methylene blue after 4 hour treatment withbuffer or titrated MICs of PlySs2 of 10×MIC, 1×MIC (16 μg/ml), 0.1×MIC,0.01×MIC and 0.001×MIC PlySs2.

FIG. 20 depicts titration analysis of catheter MRSA strain CFS 992 (JMI5381) biofilm staining with methylene blue after 4 hour treatment withbuffer or titrated MICs of PlySs2 of 10×MIC, 1×MIC (32 μg/ml), 0.1×MIC,0.01×MIC and 0.001×MIC PlySs2.

FIGS. 21A-C depict scanning electron microscopy (SEM) of 3 day oldcatheter S. aureus biofilms treated with PlySs2, washed, fixed andscanned. FIG. 21A: 0 minutes, FIG. 21B: 30 seconds and FIG. 21C: 15minutes of PlySs2 treatment are shown. 5000× magnification.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “PlySs lysin(s)”, “PlySs2 lysin”, “PlySs2” and any variantsnot specifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andpresented in FIG. 5 and SEQ ID NO: 1, and the profile of activities setforth herein and in the Claims. Accordingly, proteins displayingsubstantially equivalent or altered activity are likewise contemplated.These modifications may be deliberate, for example, such asmodifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the terms “PlySslysin(s)”, “PlySs2 lysin”, “PlySs2” are intended to include within theirscope proteins specifically recited herein as well as all substantiallyhomologous analogs, fragments or truncations, and allelic variations.PlySs2 lysin is described in U.S. Patent Application 61/477,836 and PCTApplication PCT/US2012/34456. A more recent paper Gilmer et al describesPlySs2 lysin (Gilmer D B et al (2013) Antimicrob Agents Chemother Epub2013 Apr. 9 [PMID 23571534]).

The term “ClyS”, “ClyS lysin” refers to a chimeric lysin ClyS, withactivity against Staphylococci bacteria, including Staphylococcusaureus, is detailed in WO 2010/002959 and also described in Daniel et al(Daniel, A et al (2010) Antimicrobial Agents and Chemother54(4):1603-1612). Such exemplary amino acid sequence of ClyS is providedin SEQ ID NO: 5.

A “lytic enzyme” includes any bacterial cell wall lytic enzyme thatkills one or more bacteria under suitable conditions and during arelevant time period. Examples of lytic enzymes include, withoutlimitation, various amidase cell wall lytic enzymes.

A “bacteriophage lytic enzyme” refers to a lytic enzyme extracted orisolated from a bacteriophage or a synthesized lytic enzyme with asimilar protein structure that maintains a lytic enzyme functionality.

A lytic enzyme is capable of specifically cleaving bonds that arepresent in the peptidoglycan of bacterial cells to disrupt the bacterialcell wall. It is also currently postulated that the bacterial cell wallpeptidoglycan is highly conserved among most bacteria, and cleavage ofonly a few bonds to may disrupt the bacterial cell wall. Thebacteriophage lytic enzyme may be an amidase, although other types ofenzymes are possible. Examples of lytic enzymes that cleave these bondsare muramidases, glucosaminidases, endopeptidases, orN-acetyl-muramoyl-L-alanine amidases. Fischetti et al (1974) reportedthat the C1 streptococcal phage lysin enzyme was an amidase. Garcia etal (1987, 1990) reported that the Cpl lysin from a S. pneumoniae from aCp-1 phage was a lysozyme. Caldentey and Bamford (1992) reported that alytic enzyme from the phi 6 Pseudomonas phage was an endopeptidase,splitting the peptide bridge formed by melo-diaminopemilic acid andD-alanine. The E. coli T1 and T6 phage lytic enzymes are amidases as isthe lytic enzyme from Listeria phage (ply) (Loessner et al, 1996). Thereare also other lytic enzymes known in the art that are capable ofcleaving a bacterial cell wall.

A “lytic enzyme genetically coded for by a bacteriophage” includes apolypeptide capable of killing a host bacteria, for instance by havingat least some cell wall lytic activity against the host bacteria. Thepolypeptide may have a sequence that encompasses native sequence lyticenzyme and variants thereof. The polypeptide may be isolated from avariety of sources, such as from a bacteriophage (“phage”), or preparedby recombinant or synthetic methods. The polypeptide may comprise acholine-binding portion at the carboxyl terminal side and may becharacterized by an enzyme activity capable of cleaving cell wallpeptidoglycan (such as amidase activity to act on amide bonds in thepeptidoglycan) at the amino terminal side. Lytic enzymes have beendescribed which include multiple enzyme activities, for example twoenzymatic domains, such as PlyGBS lysin.

“A native sequence phage associated lytic enzyme” includes a polypeptidehaving the same amino acid sequence as an enzyme derived from abacteria. Such native sequence enzyme can be isolated or can be producedby recombinant or synthetic means.

The term “native sequence enzyme” encompasses naturally occurring forms(e.g., alternatively spliced or altered forms) and naturally-occurringvariants of the enzyme. In one embodiment of the invention, the nativesequence enzyme is a mature or full-length polypeptide that isgenetically coded for by a gene from a bacteriophage specific forStreptococcus suis. Of course, a number of variants are possible andknown, as acknowledged in publications such as Lopez et al., MicrobialDrug Resistance 3: 199-211 (1997); Garcia et al., Gene 86: 81-88 (1990);Garcia et al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia etal., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al.,Streptococcal Genetics (J. J. Ferretti and Curtis eds., 1987); Lopez etal., FEMS Microbiol. Lett. 100: 439-448 (1992); Romero et al., J.Bacteriol. 172: 5064-5070 (1990); Ronda et al., Eur. J. Biochem. 164:621-624 (1987) and Sanchez et al., Gene 61: 13-19 (1987). The contentsof each of these references, particularly the sequence listings andassociated text that compares the sequences, including statements aboutsequence homologies, are specifically incorporated by reference in theirentireties.

“A variant sequence lytic enzyme” includes a lytic enzyme characterizedby a polypeptide sequence that is different from that of a lytic enzyme,but retains functional activity. The lytic enzyme can, in someembodiments, be genetically coded for by a bacteriophage specific forStreptococcus suis as in the case of PlySs2 having a particular aminoacid sequence identity with the lytic enzyme sequence(s) hereof, asprovided in FIG. 5 and SEQ ID NO:1. For example, in some embodiments, afunctionally active lytic enzyme can kill Streptococcus suis bacteria,and other susceptible bacteria as provided herein, including as shown inTABLE 1, 2 and 3, by disrupting the cellular wall of the bacteria. Anactive lytic enzyme may have a 60, 65, 70, 75, 80, 85, 90, 95, 97, 98,99 or 99.5% amino acid sequence identity with the lytic enzymesequence(s) hereof, as provided in FIG. 5 and in SEQ ID NO: 1. Suchphage associated lytic enzyme variants include, for instance, lyticenzyme polypeptides wherein one or more amino acid residues are added,or deleted at the N or C terminus of the sequence of the lytic enzymesequence(s) hereof, as provided in FIG. 5 and in SEQ ID NO: 1.

In a particular aspect, a phage associated lytic enzyme will have atleast about 80% or 85% amino acid sequence identity with native phageassociated lytic enzyme sequences, particularly at least about 90% (e.g.90%) amino acid sequence identity. Most particularly a phage associatedlytic enzyme variant will have at least about 95% (e.g. 95%) amino acidsequence identity with the native phage associated the lytic enzymesequence(s) hereof, as provided in FIG. 5 and in SEQ ID NO: 1 for PlySs2lysin, or as preciously described for ClyS including in WO 2010/002959and also described in Daniel et al (Daniel, A et al (2010) AntimicrobialAgents and Chemother 54(4):1603-1612).

“Percent amino acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified is defined herein as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the phage associated lyticenzyme sequence, after aligning the sequences in the same reading frameand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity.

“Percent nucleic acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the phage associated lytic enzyme sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

To determine the percent identity of two nucleotide or amino acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps may be introduced in the sequence of a first nucleotidesequence). The nucleotides or amino acids at corresponding nucleotide oramino acid positions are then compared. When a position in the firstsequence is occupied by the same nucleotide or amino acid as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=(# of identical positions/total # ofpositions)×100).

The determination of percent identity between two sequences may beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993), which is incorporated into the NBLAST program which may be usedto identify sequences having the desired identity to nucleotidesequences of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST may be utilized as described in Altschul et al.,Nucleic Acids Res, 25:3389-3402 (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,NBLAST) may be used. See the programs provided by National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health.

“Polypeptide” includes a polymer molecule comprised of multiple aminoacids joined in a linear manner. A polypeptide can, in some embodiments,correspond to molecules encoded by a polynucleotide sequence which isnaturally occurring. The polypeptide may include conservativesubstitutions where the naturally occurring amino acid is replaced byone having similar properties, where such conservative substitutions donot alter the function of the polypeptide.

The term “altered lytic enzymes” includes shuffled and/or chimeric lyticenzymes.

Phage lytic enzymes specific for bacteria infected with a specific phagehave been found to effectively and efficiently break down the cell wallof the bacterium in question. The lytic enzyme is believed to lackproteolytic enzymatic activity and is therefore non-destructive tomammalian proteins and tissues when present during the digestion of thebacterial cell wall. Furthermore, because it has been found that theaction of phage lytic enzymes, unlike antibiotics, was rather specificfor the target pathogen(s), it is likely that the normal flora willremain essentially intact (M. J. Loessner, G. Wendlinger, S. Scherer,Mol Microbiol 16, 1231-41. (1995) incorporated herein by reference). Infact, the PlySs2 lysin, while demonstrating uniquely broad bacterialspecies and strain killing, is comparatively and particularly inactiveagainst bacteria comprising the normal flora, including E. coli, asdescribed herein.

A lytic enzyme or polypeptide of use in the invention may be produced bythe bacterial organism after being infected with a particularbacteriophage or may be produced or prepared recombinantly orsynthetically as either a prophylactic treatment for preventing thosewho have been exposed to others who have the symptoms of an infectionfrom getting sick, or as a therapeutic treatment for those who havealready become ill from the infection. In as much the lysin polypeptidesequences and nucleic acids encoding the lysin polypeptides aredescribed and referenced to herein, the lytic enzyme(s)/polypeptide(s)may be preferably produced via the isolated gene for the lytic enzymefrom the phage genome, putting the gene into a transfer vector, andcloning said transfer vector into an expression system, using standardmethods of the art, including as exemplified herein. The lytic enzyme(s)or polypeptide(s) may be truncated, chimeric, shuffled or “natural,” andmay be in combination. Relevant U.S. Pat. No. 5,604,109 is incorporatedherein in its entirety by reference. An “altered” lytic enzyme can beproduced in a number of ways. In a preferred embodiment, a gene for thealtered lytic enzyme from the phage genome is put into a transfer ormovable vector, preferably a plasmid, and the plasmid is cloned into anexpression vector or expression system. The expression vector forproducing a lysin polypeptide or enzyme of the invention may be suitablefor E. coli, Bacillus, or a number of other suitable bacteria. Thevector system may also be a cell free expression system. All of thesemethods of expressing a gene or set of genes are known in the art. Thelytic enzyme may also be created by infecting Streptococcus suis with abacteriophage specific for Streptococcus suis, wherein said at least onelytic enzyme exclusively lyses the cell wall of said Streptococcus suishaving at most minimal effects on other, for example natural orcommensal, bacterial flora present (see TABLE 5, which provides theresults of lytic activity studies against various commensal human gutbacteria).

A “chimeric protein” or “fusion protein” comprises all or (preferably abiologically active) part of a polypeptide of use in the inventionoperably linked to a heterologous polypeptide. Chimeric proteins orpeptides are produced, for example, by combining two or more proteinshaving two or more active sites. Chimeric protein and peptides can actindependently on the same or different molecules, and hence have apotential to treat two or more different bacterial infections at thesame time. Chimeric proteins and peptides also may be used to treat abacterial infection by cleaving the cell wall in more than one location,thus potentially providing more rapid or effective (or synergistic)killing from a single lysin molecule or chimeric peptide.

A “heterologous” region of a DNA construct or peptide construct is anidentifiable segment of DNA within a larger DNA molecule or peptidewithin a larger peptide molecule that is not found in association withthe larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA or peptide as defined herein.

The term “operably linked” means that the polypeptide of the disclosureand the heterologous polypeptide are fused in-frame. The heterologouspolypeptide can be fused to the N-terminus or C-terminus of thepolypeptide of the disclosure. Chimeric proteins are producedenzymatically by chemical synthesis, or by recombinant DNA technology. Anumber of chimeric lytic enzymes have been produced and studied. Oneexample of a useful fusion protein is a GST fusion protein in which thepolypeptide of the disclosure is fused to the C-terminus of a GSTsequence. Such a chimeric protein can facilitate the purification of arecombinant polypeptide of the disclosure.

In another embodiment, the chimeric protein or peptide contains aheterologous signal sequence at its N-terminus. For example, the nativesignal sequence of a polypeptide of the disclosure can be removed andreplaced with a signal sequence from another known protein.

The fusion protein may combine a lysin polypeptide with a protein orpolypeptide of having a different capability, or providing an additionalcapability or added character to the lysin polypeptide. The fusionprotein may be an immunoglobulin fusion protein in which all or part ofa polypeptide of the disclosure is fused to sequences derived from amember of the immunoglobulin protein family. The immunoglobulin may bean antibody, for example an antibody directed to a surface protein orepitope of a susceptible or target bacteria. The immunoglobulin fusionprotein can alter bioavailability of a cognate ligand of a polypeptideof the disclosure. Inhibition of ligand/receptor interaction may beuseful therapeutically, both for treating bacterial-associated diseasesand disorders for modulating (i.e. promoting or inhibiting) cellsurvival. The fusion protein may include a means to direct or target thelysin, including to particular tissues or organs or to surfaces such asdevices, plastic, membranes. Chimeric and fusion proteins and peptidesof the disclosure can be produced by standard recombinant DNAtechniques.

A modified or altered form of the protein or peptides and peptidefragments, as disclosed herein, includes protein or peptides and peptidefragments that are chemically synthesized or prepared by recombinant DNAtechniques, or both. These techniques include, for example,chimerization and shuffling. As used herein, shuffled proteins orpeptides, gene products, or peptides for more than one related phageprotein or protein peptide fragments have been randomly cleaved andreassembled into a more active or specific protein. Shuffledoligonucleotides, peptides or peptide fragment molecules are selected orscreened to identify a molecule having a desired functional property.Shuffling can be used to create a protein that is more active, forinstance up to 10 to 100 fold more active than the template protein. Thetemplate protein is selected among different varieties of lysinproteins. The shuffled protein or peptides constitute, for example, oneor more binding domains and one or more catalytic domains. When theprotein or peptide is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. Accordingly such preparationsof the protein have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.

The present invention also pertains to other variants of thepolypeptides useful in the invention. Such variants may have an alteredamino acid sequence which can function as either agonists (mimetics) oras antagonists. Variants can be generated by mutagenesis, i.e., discretepoint mutation or truncation. An agonist can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the protein. An antagonist of a protein can inhibitone or more of the activities of the naturally occurring form of theprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein. Variants of a protein of use in the disclosure which functionas either agonists (mimetics) or as antagonists can be identified byscreening combinatorial libraries of mutants, such as truncationmutants, of the protein of the disclosure. In one embodiment, avariegated library of variants is generated by combinatorial mutagenesisat the nucleic acid level and is encoded by a variegated gene library.There are a variety of methods which can be used to produce libraries ofpotential variants of the polypeptides of the disclosure from adegenerate oligonucleotide sequence. Libraries of fragments of thecoding sequence of a polypeptide of the disclosure can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants, active fragments or truncations.Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. In this context, the smallest portion of a protein(or nucleic acid that encodes the protein) according to embodiments isan epitope that is recognizable as specific for the phage that makes thelysin protein. Accordingly, the smallest polypeptide (and associatednucleic acid that encodes the polypeptide) that can be expected to binda target or receptor, such as an antibody, and is useful for someembodiments may be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 75, 85, or 100 amino acids long. Although small sequences asshort as 8, 9, 10, 11, 12 or 15 amino acids long reliably compriseenough structure to act as targets or epitopes, shorter sequences of 5,6, or 7 amino acids long can exhibit target or epitopic structure insome conditions and have value in an embodiment. Thus, the smallestportion of the protein(s) or lysin polypeptides provided herein,including as set out in FIG. 5 and SEQ ID NO:1 and the domain sequencesof SEQ ID NO: 3 and 4 includes polypeptides as small as 5, 6, 7, 8, 9,10, 12, 14 or 16 amino acids long.

Biologically active portions of a protein or peptide fragment of theembodiments, as described herein, include polypeptides comprising aminoacid sequences sufficiently identical to or derived from the amino acidsequence of the lysin protein of the disclosure, which include feweramino acids than the full length protein of the lysin protein andexhibit at least one activity of the corresponding full-length protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the corresponding protein. A biologicallyactive portion of a protein or protein fragment of the disclosure can bea polypeptide which is, for example, 10, 25, 50, 100 less or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, or added can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of the native form of a polypeptide of the embodiments.

Homologous proteins and nucleic acids can be prepared that sharefunctionality with such small proteins and/or nucleic acids (or proteinand/or nucleic acid regions of larger molecules) as will be appreciatedby a skilled artisan. Such small molecules and short regions of largermolecules that may be homologous specifically are intended asembodiments. Preferably the homology of such valuable regions is atleast 50%, 65%, 75%, 80%, 85%, and preferably at least 90%, 95%, 97%,98%, or at least 99% compared to the lysin polypeptides provided herein,including as set out in FIG. 5 and SEQ ID NO: 1 and the domain sequencesof SEQ ID NO: 3 and 4. These percent homology values do not includealterations due to conservative amino acid substitutions.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, atleast about 85%, and preferably at least about 90 or 95%) are identical,or represent conservative substitutions. The sequences of comparablelysins, such as comparable PlySs2 lysins, or comparable ClyS lysins, aresubstantially homologous when one or more, or several, or up to 10%, orup to 15%, or up to 20% of the amino acids of the lysin polypeptide aresubstituted with a similar or conservative amino acid substitution, andwherein the comparable lysins have the profile of activities,anti-bacterial effects, and/or bacterial specificities of a lysin, suchas the PlySs2 lysin and/or ClyS lysin, disclosed herein.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

Mutations can be made in the amino acid sequences, or in the nucleicacid sequences encoding the polypeptides and lysins herein, including inthe lysin sequences set out in FIG. 5 and in SEQ ID NO: 1 or in thedomain sequences of SEQ ID NO: 3 or 4, or in active fragments ortruncations thereof, such that a particular codon is changed to a codonwhich codes for a different amino acid, an amino acid is substituted foranother amino acid, or one or more amino acids are deleted. Such amutation is generally made by making the fewest amino acid or nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (for example, by changing the codon from an amino acid belongingto a grouping of amino acids having a particular size or characteristicto an amino acid belonging to another grouping) or in a conservativemanner (for example, by changing the codon from an amino acid belongingto a grouping of amino acids having a particular size or characteristicto an amino acid belonging to the same grouping). Such a conservativechange generally leads to less change in the structure and function ofthe resulting protein. A non-conservative change is more likely to alterthe structure, activity or function of the resulting protein. Thepresent invention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0) Lysine, Arginine,Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Exemplary and preferred conservative amino acid substitutions includeany of:

glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) forvaline (V) and vice versa; serine (S) for threonine (T) and vice versa;isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine(Q) and vice versa; isoleucine (I) for methionine (M) and vice versa;serine (S) for asparagine (N) and vice versa; leucine (L) for methionine(M) and vice versa; lysine (L) for glutamic acid (E) and vice versa;alanine (A) for serine (S) and vice versa; tyrosine (Y) forphenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid(D) and vice versa; leucine (L) for isoleucine (I) and vice versa;lysine (K) for arginine (R) and vice versa.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Thus, one of skill in the art, based on a review of the sequence of thePlySs2 lysin polypeptide provided herein and on their knowledge and thepublic information available for other lysin polypeptides, can makeamino acid changes or substitutions in the lysin polypeptide sequence.Amino acid changes can be made to replace or substitute one or more, oneor a few, one or several, one to five, one to ten, or such other numberof amino acids in the sequence of the lysin(s) provided herein togenerate mutants or variants thereof. Such mutants or variants thereofmay be predicted for function or tested for function or capability forkilling bacteria, including Staphylococcal, Streptococcal, Listeria, orEnterococcal bacteria, and/or for having comparable activity to thelysin(s) as described and particularly provided herein. Thus, changescan be made to the sequence of lysin, and mutants or variants having achange in sequence can be tested using the assays and methods describedand exemplified herein, including in the examples. One of skill in theart, on the basis of the domain structure of the lysin(s) hereof canpredict one or more, one or several amino acids suitable forsubstitution or replacement and/or one or more amino acids which are notsuitable for substitution or replacement, including reasonableconservative or non-conservative substitutions.

In this regard, and with exemplary reference to PlySs2 lysin it ispointed out that, although the PlySs2 polypeptide lysin represents adivergent class of prophage lytic enzyme, the lysin comprises anN-terminal CHAP domain (cysteine-histidine amidohydrolase/peptidase)(SEQ ID NO: 3) and a C-terminal SH3-type 5 domain (SEQ ID NO: 4) asdepicted in FIG. 5 . The domains are depicted in the amino acid sequencein distinct shaded color regions, with the CHAP domain corresponding tothe first shaded amino acid sequence region starting with LNN . . . andthe SH3-type 5 domain corresponding to the second shaded region startingwith RSY . . . CHAP domains are included in several previouslycharacterized streptococcal and staphylococcal phage lysins. Thus, oneof skill in the art can reasonably make and test substitutions orreplacements to the CHAP domain and/or the SH-3 domain of PlySs2.Sequence comparisons to the Genbank database can be made with either orboth of the CHAP and/or SH-3 domain sequences or with the PlySs2 lysinfull amino acid sequence, for instance, to identify amino acids forsubstitution.

The PlySs2 lysin displays activity and capability to kill numerousdistinct strains and species of gram positive bacteria, includingStaphylococcal, Streptococcal, Listeria, or Enterococcal bacteria. Inparticular and with significance, PlySs2 is active in killingStaphylococcus strains, including Staphylococcus aureus, particularlyboth antibiotic-sensitive and distinct antibiotic-resistant strains.PlySs2 is also active in killing Streptococcus strains, and showsparticularly effective killing against Group A and Group B streptococcusstrains. PlySs2 lysin capability against bacteria is depicted below inTABLE 1, based on log kill assessments using isolated strains in vitro.Activity of PlySs2 against various Gram-positive and Gram-negativeorganisms and against antibiotic resistant Staphylococcus aureus strainsis tabulated below in TABLES 2 and 3. MIC ranges for PlySs2 against thebacteria is noted to provide relative killing activity.

TABLE 1 PlySs2 Reduction in Growth of Different Bacteria (partiallisting) Bacteria Relative Kill with PlySs2 Staphylococcus aureus +++(VRSA, VISA, MRSA, MSSA) Streptococcus suis +++ Staphylococcusepidermidis ++ Staphylococcus simulans +++ Lysteria monocytogenes ++Enterococcus faecalis ++ Streptococcus dysgalactiae - GBS ++Streptococcus agalactiae -GBS +++ Streptococcus pyogenes -GAS +++Streptococcus equi ++ Streptococcus sanguinis ++ Streptococcus gordonii++ Streptococcus sobrinus + Streptococcus rattus + Streptococcusoralis + Streptococcus pneumonine + Bacillus thuringiensis − Bacilluscereus − Bacillus subtilis − Bacillus anthracis − Escherichia coli −Enterococcus faecium − Pseudomanas aeruginosa −

TABLE 2 Susceptible and Non-susceptible Bacterial Strains Organism andsusceptibility subset MIC (μg/mL) (no. tested) 50% 90% RangeStaphylcoccus aureus Methicillin susceptible (103) 4 8 1-16 Methicillinresistant (120) 4 8 1-16 Streptococcus pyogenes, Group A (54) 2 80.5-8   Streptococcus agalactiae, Group B (51) 8 16 1-64 OtherGram-positive organisms Staphylococcus lugdiensis (10) 8 8   8Staphylococcus epidermidis (11) 128 512  4-512 Streptococcus pneumoniae(26) 16 64 1-64 Streptococcus mutans (12) 64 256  2-256 Listeriamonocytogenes (12) 128 512  1-512 Enterococcus faecalis (17) >512 >512 32->512 Enterococcus faecium (5) >512 >512  32->512 Bacillus cereus(10) >512 >512 >512 Gram-negative organisms Acinetobacter baumannii(8) >512 >512 >512 Escherichia coli (6) >512 >512 >512 Pseudomonasaeruginosa (5) >512 >512 >512

TABLE 3 Activity of PlySs2 Against Antibiotic- Resistant Staphylococcusaureus MIC (mg/mL) Susceptibility subset (no. tested) 50% 90% RangeVancomycin-resistant (14) 2 4 1-4 Vancomycin-intermediate (31) 8 32 1-64 Linezolid-resistant (5) 2 2 2-4 Daptomycin-resistant (8) 2 4 2-4

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show significant binding tomolecules other than its specific binding partner(s). The term is alsoapplicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is tosay permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention hereof, those of skill in the art will appreciate thatminor modifications to the N- or C-terminal of the peptide may howeverbe contemplated, such as the chemical modification of the terminal toadd a protecting group or the like, e.g. the amidation of theC-terminus.

The term “isolated” refers to the state in which the lysinpolypeptide(s) of the invention, or nucleic acid encoding suchpolypeptides will be, in accordance with the present invention.Polypeptides and nucleic acid will be free or substantially free ofmaterial with which they are naturally associated such as otherpolypeptides or nucleic acids with which they are found in their naturalenvironment, or the environment in which they are prepared (e.g. cellculture) when such preparation is by recombinant DNA technologypractised in vitro or in vivo. Polypeptides and nucleic acid may beformulated with diluents or adjuvants and still for practical purposesbe isolated—for example the polypeptides will normally be mixed withpolymers or mucoadhesives or other carriers, or will be mixed withpharmaceutically acceptable carriers or diluents, when used in diagnosisor therapy.

Nucleic acids capable of encoding the S. suis PlySs2 lysinpolypeptide(s) useful and applicable in the invention are providedherein. Representative nucleic acid sequences in this context arepolynucleotide sequences coding for the polypeptide of FIG. 5 or SEQ IDNO: 1, particularly polynucleotide sequences of SEQ ID NO: 2 capable ofencoding the polypeptide of SEQ ID NO: 1, and sequences that hybridize,under stringent conditions, with complementary sequences of the DNA ofSEQ ID NO: 2 and/or the FIG. 5 sequence(s). Further variants of thesesequences and sequences of nucleic acids that hybridize with those shownin the figures also are contemplated for use in production of lysingenzymes according to the disclosure, including natural variants that maybe obtained. A large variety of isolated nucleic acid sequences or cDNAsequences that encode phage associated lysing enzymes and partialsequences that hybridize with such gene sequences are useful forrecombinant production of the lysin enzyme(s) or polypeptide(s) of theinvention.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

DNA molecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and which differ from those disclosed bythe deletion, addition or substitution of nucleotides while stillencoding a protein which possesses the functional characteristic of thelysin polypeptide(s) are contemplated by the disclosure. Also includedare small DNA molecules which are derived from the disclosed DNAmolecules. Such small DNA molecules include oligonucleotides suitablefor use as hybridization probes or polymerase chain reaction (PCR)primers. As such, these small DNA molecules will comprise at least asegment of a lytic enzyme genetically coded for by a bacteriophage ofStaphylococcus suis and, for the purposes of PCR, will comprise at leasta 10-15 nucleotide sequence and, more preferably, a 15-30 nucleotidesequence of the gene. DNA molecules and nucleotide sequences which arederived from the disclosed DNA molecules as described above may also bedefined as DNA sequences which hybridize under stringent conditions tothe DNA sequences disclosed, or fragments thereof.

In preferred embodiments of the present disclosure, stringent conditionsmay be defined as those under which DNA molecules with more than 25%sequence variation (also termed “mismatch”) will not hybridize. In amore preferred embodiment, stringent conditions are those under whichDNA molecules with more than 15% mismatch will not hybridize, and morepreferably still, stringent conditions are those under which DNAsequences with more than 10% mismatch will not hybridize. Preferably,stringent conditions are those under which DNA sequences with more than6% mismatch will not hybridize.

The degeneracy of the genetic code further widens the scope of theembodiments as it enables major variations in the nucleotide sequence ofa DNA molecule while maintaining the amino acid sequence of the encodedprotein. Thus, the nucleotide sequence of the gene could be changed atthis position to any of these three codons without affecting the aminoacid composition of the encoded protein or the characteristics of theprotein. The genetic code and variations in nucleotide codons forparticular amino acids are well known to the skilled artisan. Based uponthe degeneracy of the genetic code, variant DNA molecules may be derivedfrom the cDNA molecules disclosed herein using standard DNA mutagenesistechniques as described above, or by synthesis of DNA sequences. DNAsequences which do not hybridize under stringent conditions to the cDNAsequences disclosed by virtue of sequence variation based on thedegeneracy of the genetic code are herein comprehended by thisdisclosure.

Thus, it should be appreciated that also within the scope of the presentinvention are DNA sequences encoding a lysin of the present invention,including PlySs2 and PlySs1, which sequences code for a polypeptidehaving the same amino acid sequence as provided in FIG. 5 or SEQ ID NO:1, but which are degenerate thereto or are degenerate to the exemplarynucleic acids sequences provided in FIG. 5 and in SEQ ID NO: 2. By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art the codonswhich can be used interchangeably to code for each specific amino acid.

One skilled in the art will recognize that the DNA mutagenesistechniques described here and known in the art can produce a widevariety of DNA molecules that code for a bacteriophage lysin ofStreptococcus suis yet that maintain the essential characteristics ofthe lytic polypeptides described and provided herein. Newly derivedproteins may also be selected in order to obtain variations on thecharacteristic of the lytic polypeptide(s), as will be more fullydescribed below. Such derivatives include those with variations in aminoacid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation may bepredetermined, the mutation per se does not need to be predetermined.Amino acid substitutions are typically of single residues, or can be ofone or more, one or a few, one, two, three, four, five, six or sevenresidues; insertions usually will be on the order of about from 1 to 10amino acid residues; and deletions will range about from 1 to 30residues. Deletions or insertions may be in single form, but preferablyare made in adjacent pairs, i.e., a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. Substitutionvariants are those in which at least one residue in the amino acidsequence has been removed and a different residue inserted in its place.Such substitutions may be made so as to generate no significant effecton the protein characteristics or when it is desired to finely modulatethe characteristics of the protein. Amino acids which may be substitutedfor an original amino acid in a protein and which are regarded asconservative substitutions are described above and will be recognized byone of skill in the art.

As is well known in the art, DNA sequences may be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host. Such operative linking of aDNA sequence of this invention to an expression control sequence, ofcourse, includes, if not already part of the DNA sequence, the provisionof an initiation codon, ATG, in the correct reading frame upstream ofthe DNA sequence. A wide variety of host/expression vector combinationsmay be employed in expressing the DNA sequences of this invention.Useful expression vectors, for example, may consist of segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Any of a widevariety of expression control sequences—sequences that control theexpression of a DNA sequence operatively linked to it—may be used inthese vectors to express the DNA sequences of this invention. A widevariety of unicellular host cells are also useful in expressing the DNAsequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, human cells and plant cells in tissue culture. One skilled in theart will be able to select the proper vectors, expression controlsequences, and hosts without undue experimentation to accomplish thedesired expression without departing from the scope of this invention.

As used herein and referred to in the art, a biofilm is an aggregate ofmicrobes with a distinct architecture. Biofilm formation involvesattachment of free floating microorganisms to a surface. A biofilm isessentially a collective in which microbial cells, each only amicrometer or two long, form convoluted structures, including towersthat can be hundreds of micrometers high. The channels within biofilmsact as fluid-filled conduits that circulate nutrients, oxygen, wasteproducts, etc., as required to maintain a viable biofilm community. Thebiofilm or microbial (bacterial, fungal, or algal) community istypically enveloped by extracellular biopolymers produced by themicrobial cells and adheres to the interface between a liquid andsurface. The encapsulated property of biofilms is one of severalfeatures that renders the microbial organisms therein highly resistantto standard anti-microbial therapeutics. Bacteria growing in a biofilm,for example, are highly resistant to antibiotics, and in some cases areup to 1,000 times more resistant than the same bacteria growing withouta biofilm superstructure.

Standard antibiotic therapy can be useless wherein a biofilmcontaminated implant is detected and the only recourse under suchcircumstances may be to remove the contaminated implant. Biofilms are,furthermore, involved in numerous chronic diseases. Cystic fibrosispatients, for example, suffer from Pseudomonas infections that oftenresult in antibiotic resistant biofilms. Biofilm formation occurs whenfree floating microorganisms attach themselves to a surface. Becausebiofilms protect the bacteria, they are often more resistant totraditional antimicrobial treatments, making them a serious health risk,which is evidenced by more than one million cases of catheter-associatedurinary tract infections (CAUTI) reported each year, many of which canbe attributed to biofilm-associated bacteria (Donlan, R M (2001) EmergInfect Dis 7(2):277-281; Maki D and Tambyah P (2001) Emerg Infect Dis7(2):342-347)

Various approaches have been attempted to prevent biofilm formation andinclude inhibiting protein adsorption or biofilm adhesion using chemicaland mechanical means. Chemical approaches include antimicrobial coatingson indwelling devices and polymer modifications. Antibiotics, biocides,and ion coatings are examples of chemical methods of biofilm preventionand may interfere with the attachment and expansion of immaturebiofilms. However, these coatings are effective only for a short timeperiod (about 1 week), after which leaching of the antimicrobial agentreduces the effectiveness of the coating (Dror N et al (2009) Sensors9(4):2538-2554). Several in vitro studies have confirmed theeffectiveness of silver at preventing infection, both in coating formand as nanoparticles dispersed in a polymer matrix. However, concernsremain over the use of silver in vivo with potential toxic effects onhuman tissue and there has been limited use of silver coatings. Despitethis, silver coatings are used on devices such as catheters (Vasilev Ket al (2009) Expert Rev Med Devices 6(5):553-567). Via polymermodification, antimicrobial agents can be immobilized on device surfacesusing long, flexible polymeric chains. These chains are anchored to thedevice surface by covalent bonds, producing non-leaching,contact-killing surfaces. An in vitro study found that whenN-alkylpyridinium bromide, an antimicrobial agent, was attached to apoly(4-vinyl-N-hexylpyridine), the polymer was capable of inactivatingmore than 99% of S. epidermidis, E. coli, and P. aeruginosa bacteria(Jansen B and Kohnen W (1995) J Ind Microbiol 15(4):391-396).

Mechanical approaches to preventing biofilms include altering thesurface of devices such as catheters, including modifying thehydrophobicity of the device surface, altering its physical nature usingsmooth-surfaced materials, and altering surface charge. Thehydrophobicity and the charge of polymeric chains can be controlled byusing several backbone compounds and antimicrobial agents, includingpositively charged polycations. In another approach, low-energy surfaceacoustic waves are produced from a battery powered device that deliversperiodic rectangular pulses and waves spread to the surface, in thiscase a catheter, creating horizontal waves that prevent the adhesion ofbacteria to surfaces. This technique has been tested on white rabbitsand guinea pigs and lowered biofilm growth (Hazan, Z et al (2006)Antimicrob Agents and Chemother 50(12):4144-152).

In accordance with the present invention, methods and compositions areprovided for prevention, dispersion and treatment of bacterial biofilms.Methods and compositions are particularly provided for prevention,dispersion and treatment of biofilms comprising Staphylococcal bacteria.In particular, methods and compositions for prevention, dispersion andtreatment of biofilms comprising Staphylococcus aureus, including orcomprising antibiotic-resistant and/or antibiotic-sensitive S. aureusare an aspect of the invention. In an aspect of the invention, themethods and compositions of the invention comprise a lysin, particularlyPlySs2 lysin, which is capable of killing Staphylococcal andStreptococcal bacteria, including antibiotic-resistant bacteria.

The methods and compositions of the invention, particularly comprisingPlySs2 lysin, may be combined or incorporated with chemical ormechanical means, compositions or approaches for prevention ordispersion of biofilms. Thus, the compositions herein may be combined orincorporated with antibiotics, biocides, and ion coatings in minimizingthe growth or establishment of biofilms, particularly in or onin-dwelling devices or catheters. By way of example and not limitation,a composition comprising PlySs2 may be administered or otherwiseprovided in presterilizing or maintaining an indwelling device orcatheter biofilm free or with reduced bacterial adhesion or reduced riskof biofilm formation. Thus, a composition comprising PlySs2 may beutilized in solution to flush or regularly clean and maintain anindwelling device, catheter, etc biofilm free or with reduced bacterialadhesion or reduced risk of biofilm formation. In an instance where abiofilm is suspected, evident, or demonstrated, a composition comprisingPlySs2 may be administered or otherwise contacted with the biofilm orthe device, region, location, site so as to facilitate, initiate, orresult in dispersion, alleviation, removal, or treatment of the biofilm.Thus, for example, in instances wherein a patient presents with elevatedtemperature, or with discomfort, redness, swelling associated witharound a device or catheter, a composition comprising PlySs2 may beadministered to the patient or contacted with the device or catheter toalleviate, dispel or treat the relevant temperature, discomfort,redness, swelling by dispersing, preventing or treating any biofilmbeing formed or having formed.

In accordance with the invention, a composition comprising lysin,particularly PlySs2 lysin or active variants thereof, may beadministered or otherwise contacted with an established or suspectedbiofilm or the device, region, location, site with biofilm, in a singleor in multiple doses or administrations. The lysin may be administeredalong with, before, or after one or more antibiotic. The lysin may beadministered in an initial dose, for example, followed by or along withantibiotic, and the initial dose of lysin may be followed by asubsequent dose of lysin. In one such situation, the initial dose oflysin, particularly PlySs2, may serve to disperse the biofilm, followedby a subsequent dose of lysin (of lower, same or higher amount, whichmay depend in part on the initial response and dispersion of thebiofilm) which may serve to further disperse or additionally kill ordecolonize the bacteria in or of or from the biofilm. A dose ofantibiotic may be administered also subsequently or in addition tofurther serve to disperse or additionally kill or decolonize thebacteria in or of or from the biofilm.

Therapeutic or pharmaceutical compositions comprising the lyticenzyme(s)/polypeptide(s) of use in the methods and applications providedin the invention are provided herein, as well as related methods of use.Therapeutic or pharmaceutical compositions may comprise one or morelytic polypeptide(s), and optionally include natural, truncated,chimeric or shuffled lytic enzymes, optionally combined with othercomponents such as a carrier, vehicle, polypeptide, polynucleotide,holin protein(s), one or more antibiotics or suitable excipients,carriers or vehicles. The invention provides therapeutic compositions orpharmaceutical compositions of the lysins of the invention, includingPlySs2 for use in the killing, alleviation, decolonization, prophylaxisor treatment of gram-positive bacteria in biofilms and particularly fordispersing, preventing or treating biofilms.

The enzyme(s) or polypeptide(s) included in the therapeutic compositionsof use in the method of the invention may be one or more or anycombination of unaltered phage associated lytic enzyme(s), truncatedlytic polypeptides, variant lytic polypeptide(s), and chimeric and/orshuffled lytic enzymes. Additionally, different lytic polypeptide(s)genetically coded for by different phage for treatment of the samebacteria may be used. These lytic enzymes may also be any combination of“unaltered” lytic enzymes or polypeptides, truncated lyticpolypeptide(s), variant lytic polypeptide(s), and chimeric and shuffledlytic enzymes. The lytic enzyme(s)/polypeptide(s) in a therapeutic orpharmaceutical composition for gram-positive bacteria, includingStreptococcus, Staphylococcus, Enterococcus and Listeria, may be usedalone or in combination with antibiotics or, if there are other invasivebacterial organisms to be treated, in combination with other phageassociated lytic enzymes specific for other bacteria being targeted. Thelytic enzyme, truncated enzyme, variant enzyme, chimeric enzyme, and/orshuffled lytic enzyme may be used in conjunction with a holin protein.The amount of the holin protein may also be varied. Various antibioticsmay be optionally included in the therapeutic composition with theenzyme(s) or polypeptide(s) and with or without the presence oflysostaphin. More than one lytic enzyme or polypeptide may be includedin the therapeutic composition.

The pharmaceutical composition of use in the method of the invention canalso include one or more altered lytic enzymes, including isozymes,analogs, or variants thereof, produced by chemical synthesis or DNArecombinant techniques. In particular, altered lytic protein can beproduced by amino acid substitution, deletion, truncation,chimerization, shuffling, or combinations thereof. The pharmaceuticalcomposition may contain a combination of one or more natural lyticprotein and one or more truncated, variant, chimeric or shuffled lyticprotein. The pharmaceutical composition may also contain a peptide or apeptide fragment of at least one lytic protein derived from the same ordifferent bacteria species, with an optional addition of one or morecomplementary agent, and a pharmaceutically acceptable carrier ordiluent.

The pharmaceutical composition of use in the present methods can containa complementary agent, including one or more antimicrobial agent and/orone or more conventional antibiotics, particularly as provided herein.In order to accelerate treatment of the infection or dispersion of thebacterial biofilm, the therapeutic agent may further include at leastone complementary agent which can also potentiate the bactericidalactivity of the lytic enzyme. Antimicrobials act largely by interferingwith the structure or function of a bacterial cell by inhibition of cellwall synthesis, inhibition of cell-membrane function and/or inhibitionof metabolic functions, including protein and DNA synthesis. Antibioticscan be subgrouped broadly into those affecting cell wall peptidoglycanbiosynthesis and those affecting DNA or protein synthesis in grampositive bacteria. Cell wall synthesis inhibitors, including penicillinand antibiotics like it, disrupt the rigid outer cell wall so that therelatively unsupported cell swells and eventually ruptures. Thecomplementary agent may be an antibiotic, such as erythromycin,clarithromycin, azithromycin, roxithromycin, other members of themacrolide family, penicillins, cephalosporins, and any combinationsthereof in amounts which are effective to synergistically enhance thetherapeutic effect of the lytic enzyme. Virtually any other antibioticmay be used with the altered and/or unaltered lytic enzyme. Antibioticsaffecting cell wall peptidoglycan biosynthesis include: Glycopeptides,which inhibit peptidoglycan synthesis by preventing the incorporation ofN-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptidesubunits into the peptidoglycan matrix. Available glycopeptides includevancomycin and teicoplanin; Penicillins, which act by inhibiting theformation of peptidoglycan cross-links. The functional group ofpenicillins, the β-lactam moiety, binds and inhibits DD-transpeptidasethat links the peptidoglycan molecules in bacteria. Hydrolytic enzymescontinue to break down the cell wall, causing cytolysis or death due toosmotic pressure. Common penicillins include oxacillin, ampicillin andcloxacillin; and Polypeptides, which interfere with thedephosphorylation of the C₅₅-isoprenyl pyrophosphate, a molecule thatcarries peptidoglycan building-blocks outside of the plasma membrane. Acell wall-impacting polypeptide is bacitracin. Other useful and relevantantibiotics include vancomycin, linezolid, and daptomycin.

Similarly, other lytic enzymes may be included in the carrier to treator disperse other bacteria or bacterial infections. The pharmaceuticalcomposition can also contain a peptide or a peptide fragment of at leastone lytic protein, one holin protein, or at least one holin and onelytic protein, which lytic and holin proteins are each derived from thesame or different bacteria species, with an optional addition of one ormore complementary agent(s), and a suitable carrier or diluent.

Also of use in the methods are compositions containing nucleic acidmolecules that, either alone or in combination with other nucleic acidmolecules, are capable of expressing an effective amount of a lyticpolypeptide(s) or a peptide fragment of a lytic polypeptide(s) in vivo.Cell cultures containing these nucleic acid molecules, polynucleotides,and vectors carrying and expressing these molecules in vitro or in vivo,are also provided.

The present methods may utilize therapeutic or pharmaceuticalcompositions that comprise lytic polypeptide(s) combined with a varietyof carriers to disperse or decolonize the bacteria or treat theillnesses caused by the susceptible gram-positive bacteria. The carriersuitably contains minor amounts of additives such as substances thatenhance isotonicity and chemical stability. Such materials are non-toxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, succinate, acetic acid, and otherorganic acids or their salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) polypeptides, e.g.,polyarginine or tripeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;glycine; amino acids such as glutamic acid, aspartic acid, histidine, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, mannose, trehalose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; counter-ions such as sodium; non-ionic surfactants such aspolysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutralsalts. Glycerin or glycerol (1,2,3-propanetriol) is commerciallyavailable for pharmaceutical use. DMSO is an aprotic solvent with aremarkable ability to enhance penetration of many locally applied drugs.The carrier vehicle may also include Ringer's solution, a bufferedsolution, and dextrose solution, particularly when an intravenoussolution is prepared.

A lytic polypeptide(s) may be added to these substances in a liquid formor in a lyophilized state, whereupon it will be solubilized when itmeets body fluids such as saliva. The polypeptide(s)/enzyme may also bein a micelle or liposome.

The effective dosage rates or amounts of an altered or unaltered lyticenzyme/polypeptide(s) of and for use in the present invention willdepend in part on whether the lytic enzyme/polypeptide(s) will be usedtherapeutically or prophylactically, the duration of exposure of therecipient to the infectious bacteria, the size and weight of theindividual, etc. The duration for use of the composition containing theenzyme/polypeptide(s) also depends on whether the use is forprophylactic purposes, wherein the use may be hourly, daily or weekly,for a short time period, or whether the use will be for therapeuticpurposes wherein a more intensive regimen of the use of the compositionmay be needed, such that usage may last for hours, days or weeks, and/oron a daily basis, or at timed intervals during the day. Any dosage formemployed should provide for a minimum number of units for a minimumamount of time. Carriers that are classified as “long” or “slow” releasecarriers (such as, for example, certain nasal sprays or lozenges) couldpossess or provide a lower concentration of active (enzyme) units perml, but over a longer period of time, whereas a “short” or “fast”release carrier (such as, for example, a gargle) could possess orprovide a high concentration of active (enzyme) units per ml, but over ashorter period of time. The amount of active units per ml and theduration of time of exposure depend on the nature of infection, whethertreatment is to be prophylactic or therapeutic, and other variables.There are situations where it may be necessary to have a much higherunit/ml dosage or a lower unit/ml dosage.

The lytic enzyme/polypeptide(s) for use should be in an environmenthaving a pH which allows for activity of the lyticenzyme/polypeptide(s). A stabilizing buffer may allow for the optimumactivity of the lysin enzyme/polypeptide(s). The buffer may contain areducing reagent, such as dithiothreitol or beta mercaptoethanol (BME).The stabilizing buffer may also be or include a metal chelating reagent,such as ethylenediaminetetracetic acid disodium salt, or it may alsocontain a phosphate or citrate-phosphate buffer, or any other buffer.

A mild surfactant can be included in a therapeutic or pharmaceuticalcomposition for use in the methods in an amount effective to potentiatethe therapeutic effect of the lytic enzyme/polypeptide(s) may be used ina composition. Suitable mild surfactants include, inter alia, esters ofpolyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxypolyethoxy ethanol (Triton-X series), n-Octyl-.beta.-D-glucopyranoside,n-Octyl-.beta.-D-thioglucopyranoside, n-Decyl-.beta.-D-glucopyranoside,n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurringsurfactants, e.g., fatty acids, glycerides, monoglycerides, deoxycholateand esters of deoxycholate.

Preservatives may also be used in this invention and preferably compriseabout 0.05% to 0.5% by weight of the total composition. The use ofpreservatives assures that if the product is microbially contaminated,the formulation will prevent or diminish microorganism growth. Somepreservatives useful in this invention include methylparaben,propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDMHydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate,chlorhexidine digluconate, or a combination thereof.

The therapeutic composition of use in the present methods andapplications may further comprise other enzymes, such as the enzymelysostaphin for the treatment of any Staphylococcus aureus bacteriapresent along with the susceptible gram-positive bacteria. Lysostaphin,a gene product of Staphylococcus simulans, exerts a bacteriostatic andbactericidal effect upon S. aureus by enzymatically degrading thepolyglycine crosslinks of the cell wall (Browder et al., Res. Comm., 19:393-400 (1965)). The gene for lysostaphin has subsequently been clonedand sequenced (Recsei et al., Proc. Natl. Acad. Sci. USA, 84: 1127-1131(1987). A therapeutic composition may also include mutanolysin, andlysozyme.

Means of application of the therapeutic composition comprising a lyticenzyme/polypeptide(s) in accordance with the present methods include,but are not limited to direct, indirect, carrier and special means orany combination of means. Direct application of the lyticenzyme/polypeptide(s) may be by any suitable means to directly bring thepolypeptide in contact with the site of biofilm, infection or bacterialcolonization, such as to the nasal area (for example nasal sprays),dermal or skin applications (for example topical ointments orformulations), suppositories, tampon applications, etc. Nasalapplications include for instance nasal sprays, nasal drops, nasalointments, nasal washes, nasal injections, nasal packings, bronchialsprays and inhalers, or indirectly through use of throat lozenges,mouthwashes or gargles, or through the use of ointments applied to thenasal nares, or the face or any combination of these and similar methodsof application. The forms in which the lytic enzyme may be administeredinclude but are not limited to lozenges, troches, candies, injectants,chewing gums, tablets, powders, sprays, liquids, ointments, andaerosols.

The mode of application for the lytic enzyme includes a number ofdifferent types and combinations of carriers which include, but are notlimited to an aqueous liquid, an alcohol base liquid, a water solublegel, a lotion, an ointment, a nonaqueous liquid base, a mineral oilbase, a blend of mineral oil and petrolatum, lanolin, liposomes, proteincarriers such as serum albumin or gelatin, powdered cellulose carmel,and combinations thereof. A mode of delivery of the carrier containingthe therapeutic agent includes, but is not limited to a smear, spray, atime-release patch, a liquid absorbed wipe, and combinations thereof.The lytic enzyme may be applied to a bandage either directly or in oneof the other carriers. The bandages may be sold damp or dry, wherein theenzyme is in a lyophilized form on the bandage. This method ofapplication is most effective for the treatment of infected skin. Thecarriers of topical compositions may comprise semi-solid and gel-likevehicles that include a polymer thickener, water, preservatives, activesurfactants or emulsifiers, antioxidants, sun screens, and a solvent ormixed solvent system Polymer thickeners that may be used include thoseknown to one skilled in the art, such as hydrophilic and hydroalcoholicgelling agents frequently used in the cosmetic and pharmaceuticalindustries. Other preferred gelling polymers includehydroxyethylcellulose, cellulose gum, MVE/MA decadiene crosspolymer,PVM/MA copolymer, or a combination thereof.

It may be advantageous to have materials which exhibit adhesion tomucosal tissues, to be administered with one or more phage enzymes andother complementary agents over a period of time. Materials havingcontrolled release capability are particularly desirable, and the use ofsustained release mucoadhesives has received a significant degree ofattention. Other approaches involving mucoadhesives which are thecombination of hydrophilic and hydrophobic materials, are known.Micelles and multilamillar micelles may also be used to control therelease of enzyme. Materials having capacity to target or adhere tosurfaces, such as plastic, membranes, devices utilized in clinicalpractice, including particularly any material or component which isplaced in the body and susceptible to bacterial adhesion or biofilmdevelopment, such as catheters, valves, prosthetic devices, drug orcompound pumps, stents, orthopedic materials, etc, may be combined,mixed, or fused to the lysin(s) of use in the present invention.

Therapeutic or pharmaceutical compositions of use in the method can alsocontain polymeric mucoadhesives including a graft copolymer comprising ahydrophilic main chain and hydrophobic graft chains for controlledrelease of biologically active agents. The compositions of thisapplication may optionally contain other polymeric materials, such aspoly(acrylic acid), poly,-(vinyl pyrrolidone), and sodium carboxymethylcellulose plasticizers, and other pharmaceutically acceptable excipientsin amounts that do not cause deleterious effect upon mucoadhesivity ofthe composition.

A lytic enzyme/polypeptide(s) of the invention may be administered foruse in accordance with the invention by any pharmaceutically applicableor acceptable means including topically, orally or parenterally. Forexample, the lytic enzyme/polypeptide(s) can be administeredintramuscularly, intrathecally, subdermally, subcutaneously, orintravenously to treat infections by gram-positive bacteria. In caseswhere parenteral injection is the chosen mode of administration, anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.A vasoconstriction agent can be added to the formulation. Thepharmaceutical preparations according to this application are providedsterile and pyrogen free.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The effective dosage rates or amounts of the lytic enzyme/polypeptide(s)to be administered, and the duration of treatment will depend in part onthe seriousness of the infection, the weight of the patient,particularly human, the duration of exposure of the recipient to theinfectious bacteria, the number of square centimeters of skin or tissueor surface which are infected, the depth of the infection, theseriousness of the infection, and a variety of a number of othervariables. The composition may be applied anywhere from once to severaltimes a day, week, month, and may be applied for a short, such as daysor up to several weeks, or long term period, such as many weeks or up tomonths. The usage may last for days or weeks or longer. Any dosage formemployed should provide for a minimum number of units for a minimumamount of time. The concentration of the active units of enzymesbelieved to provide for an effective amount or dosage of enzymes may beselected as appropriate.

The lysin may be administered in a single dose or multiple doses, singlyor in combination with another agent, such as one or more antibiotic.The lysin, optionally with another agent, such as antibiotic, may beadministered by the same mode of administration or by different modes ofadministration. The lysin may be administered once, twice or multipletimes, one or more in combination or individually. Thus, lysin may beadministered in an initial dose followed by a subsequent dose or doses,particularly depending on the response and bacterial killing ordecolonization or the dispersion of the biofilm or killing of bacteriain the biofilm, and may be combined or alternated with antibioticdose(s). In a particular aspect of the invention a lysin, particularlyPlySs2, or combinations of antibiotic and lysin may be administered forlonger periods and dosing can be extended without risk of resistance.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds, addedadditional compound(s), or lysin enzyme compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term ‘assay’ means any process used to measure a specific propertyof a compound. A ‘screening assay’ means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

‘Therapeutically effective amount’ means that amount of a drug,compound, antimicrobial, antibody, polypeptide, or pharmaceutical agentthat will elicit the biological or medical response of a subject that isbeing sought by a medical doctor or other clinician. In particular, withregard to gram-positive bacterial infections and growth of gram-positivebacteria, the term “effective amount” is intended to include aneffective amount of a compound or agent that will bring about abiologically meaningful decrease in the amount of or extent of infectionof gram-positive bacteria, including having a bacteriocidal and/orbacteriostatic effect. The phrase “therapeutically effective amount” isused herein to mean an amount sufficient to prevent, and preferablyreduce by at least about 30 percent, more preferably by at least 50percent, most preferably by at least 90 percent, a clinicallysignificant change in the growth or amount of infectious bacteria, orother feature of pathology such as for example, elevated fever or whitecell count as may attend its presence and activity.

The term ‘treating’ or ‘treatment’ of any disease or infection refers,in one embodiment, to ameliorating the disease or infection (i.e.,arresting the disease or growth of the infectious agent or bacteria orreducing the manifestation, extent or severity of at least one of theclinical symptoms thereof). In another embodiment ‘treating’ or‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or infection,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, ‘treating’ or ‘treatment’ relates to slowingthe progression of a disease or reducing an infection.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

It is noted that in the context of treatment methods which are carriedout in vivo or medical and clinical treatment methods in accordance withthe present application and claims, the term subject, patient orindividual is intended to refer to a human.

The terms “gram-positive bacteria”, “Gram-positive bacteria”,“gram-positive” and any variants not specifically listed, may be usedherein interchangeably, and as used throughout the present applicationand claims refer to Gram-positive bacteria which are known and/or can beidentified by the presence of certain cell wall and/or cell membranecharacteristics and/or by staining with Gram stain. Gram positivebacteria are known and can readily be identified and may be selectedfrom but are not limited to the genera Listeria, Staphylococcus,Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, andClostridium, and include any and all recognized or unrecognized speciesor strains thereof. In an aspect of the invention, the PlyS lysinsensitive gram-positive bacteria include bacteria selected from one ormore of Listeria, Staphylococcus, Streptococcus, and Enterococcus.

The term “bacteriocidal” refers to capable of killing bacterial cells.

The term “bacteriostatic” refers to capable of inhibiting bacterialgrowth, including inhibiting growing bacterial cells.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a target cellular mass, or other feature of pathology suchas for example, elevated blood pressure, fever or white cell count asmay attend its presence and activity.

The invention provides methods for the prevention, dispersion, treatmentand/or decolonization of bacterial biofilms and the prevention ofinfections after dispersion of biofilm(s) wherein one or more grampositive bacteria, particularly one or more of Staphylococcus,Streptococcus, Enterococcus and Listeria bacteria, is suspected orpresent, comprising administering lysin, particularly PlySs2 lysin,having capability to kill S. aureus bacteria including MRSA. Theinvention provides methods for reducing or preventing biofilm growth onthe surface of devices, implants, separation membranes (for example,pervaporation, dialysis, reverse osmosis, ultrafiltration, andmicrofiltration membranes) comprising administering or utilizing lysin,particularly PlySs2 lysin, having capability to kill S. aureus bacteriaincluding MRSA.

The invention provides a method for treating a catheter-associatedurinary tract infection (CAUTI), wherein the infection is attributed tobiofilm-associated bacteria, by administering a composition comprisingPlySs2 lysin. The invention provides compositions comprising PlySs2lysin for use in treating a catheter-associated urinary tract infection(CAUTI), wherein the infection is attributed to biofilm-associatedbacteria. The methods or compositions comprise PlySs2 lysin, includingthe polypeptide as provided in FIG. 5 or SEQ ID NO: 1 or variantsthereof capable of killing Staphylococcal and Streptococcal bacteria,including S. aureus. The methods or compositions may additionallycomprise one or more antibiotic.

Endocarditis, including Staphylococcal endocarditis in the heart, suchas in an aortic valve or other valve or stent or device implanted in theheart or vessels thereof, is a significant clinical concern, risk andreality for many heart patients. The invention provides a method forreducing, preventing, dispersing or treating endocarditis, includingStaphylococcal endocarditis, and for prevention or treatment ofbiofilm(s) on heart valves or vascular stents. In these methods lysin,particularly PlySs2 lysin or active variants thereof as provided herein,is administered to prevent or treat Staphylococcal endocarditis orbiofilm(s) on heart valves or vascular stents.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1

PlySs2 lysin demonstrates the ability to kill various strains ofclinically significant gram positive bacteria, including methicillin andvancomycin resistant and sensitive strains of Staphylococcus aureus(MRSA, MSSA, VRSA and VISA). PlySs2 is a unique lysin in having broadspecies killing activity and can kill multiple species of bacteria,particularly gram-positive bacteria, significantly variousantibiotic-sensitive and antibiotic-resistant Staphylococcus, and alsoStreptococcus, including Group A and Group B streptococcus. Other PlySs2sensitive bacteria include Enterococcus and Listeria bacterial strains.A tabulation of sensitivity of various bacteria, including staphylococciand streptococci, to PlySs2 lysin is provided above including in TABLES2 and 3.

A tabulation of additional MIC studies is shown below in TABLE 4.

TABLE 4 PlySs2 and antibiotic activity against S. aureus strains*Organisms PlySs2 Daptomycin Vancomycin Oxacillin Linezolid (#of strains)MIC₉₀ [uM] MIC₉₀ [uM] MIC₉₀ [uM] MIC_(50/90) [uM] MIC_(50/90) [uM] MRSA(n = 45) 4 0.15 1 0.6 1 0.7 >4* >10.0 2 5.7 MSSA (n-28) 4 0.15 1 0.6 10.7 n/a n/a 2 5.7 VISA (n = 10) 32 1.2 8 4.9 4 2.7 n/a n/a 2 5.7 VRSA (n= 14) 2 0.08 1 0.6 >16 >10.6 n/a n/a 2 5.7 LRSA (n = 5) 2 0.08 1 0.6 10.7 n/a n/a >64 >183 DRSA (n = 8) 4 0.15 16 9.9 1 0.7 n/a n/a 2 5.7*MICs were determined using the broth microdilution method andevaluating 80% growth inhibition according to CLSI methods (M07-A9).*Red/Bold = drug failure (MIC value is above EUCAST breakpoint for theindicated drug on S. aureus)

Notably and uniquely, despite activity against numerous clinicallysignificant bacteria, including numerous Staphylococcus andStreptococcus strains and others tested as indicated in the aboveTables, PlySs2 displays at most only minimal effects on other bacteria,particularly natural or commensal bacterial flora. TABLE 5 belowdemonstrates little lytic activity of PlySs2 against various commensalhuman gut bacteria.

TABLE 5 Sensitivity of Gut Bacteria to PlySs2 Organism N (# tested)CF-301 MIC (ug/ml) Salmonella enteriditis 1 >512 Pseudomonas aeruginosa11 >512 Escherichia coli 10 >512 Klebsiella spp. 8 >512 Proteusmirabilis 2 >512 Lactobacillus spp. 6 >512 Lactococcus spp. 3 >512

Biofilm formation is a key feature in the pathogenesis of many bacterialinfections (31). Within infected tissues (i.e. heart valves inendocarditis or bone in osteomyelitis) or on implants (i.e. replacementjoints and catheters), bacterial pathogens such as S. aureus exist inbiofilms providing a favorable environment for growth and persistence,protected from the action of antibiotics and the immune system (32). Thestudies provided herein now demonstrate the potent anti-biofilm activityof PlySs2 lysin at only a 1×MIC concentration, in comparison to thecomplete inactivity of antibiotics used at 1000×MIC concentrations. Thispotent lysin anti-biofilm activity provides a means and compositionswhich are effective against biofilms and will uniquely complement theaction of antibiotics by enabling access to lysin disrupted biofilms.

In view of PlySs2's rapid bacterial killing and effects on numerousclinically significant bacterial strains and species, the efficacy ofPlySs2 lysin against Staphylococcus aureus biofilms was tested in vitrousing biofilm assays.

Minimally inhibitory concentration of PlySs2 lysin against methicillinresistant S. aureus MRSA strain ATCC BAA-42 was determined as 16 μg/ml.This value is the MIC determined in the presence of reducing agent (suchas DTT or BMS) in the MIC assay. Reducing agent is added for the purposeof improving reproducibility between and among assays in determining MICvalues. Biofilm studies are conducted without added reducing agent. TheMIC value for BAA-42 in the absence of reducing agent is 32 μg/ml. TheMIC value is consistent with other MRSA strains on average as noted inthe tables provided above (see Tables 2 and 4). MICs were determinedusing the broth microdilution method in accordance with standards and asdescribed in the Clinical and Laboratory Standards Institute (CLSI)document M07-A9 (Methods for dilutional antimicrobial sensitivity testsfor bacteria that grow aerobically. Volume 32 (Wayne [PA]: Clinical andLaboratory Standards Institute [US], 2012).

Biofilms were generated using a variation of the method described by Wuet al (Wu J A et al (2003) Antimicrob Agents and Chemother47(11):3407-3414). Briefly, 1×10⁶ stationary phase cells ofmethicillin-resistant S. aureus (MRSA) strain ATCC BAA-42 wereinoculated into 2 ml of tryptic-soy broth supplemented with 1% glucoseand grown for 18 hours in 24-well tissue culture dishes at 37° C.without aeration. Planktonic cells (non-adherent bacteria) were removedby washing with 1×PBS and remaining bacteria (sessile, or biofilmbacteria) were then treated with the with PlySs2 lysin or withantibiotic (daptomycin, linezolid or vancomycin obtained fromSigma-Aldrich) at various concentrations for up to 24 hours. At thevarious time points (0 hours, 2 hours, 4 hours, up to 24 hours), thewells were washed withIX PBS, fixed by air-drying at 37° C. for 15minutes, and stained with 1 ml of 1% crystal violet solution(Sigma-Aldrich) To visualize remaining biofilm. The optical density ofbiofilms stained with crystal violet was also determined to provide amore quantitative comparison. An exemplary density study is provided inFIG. 7 .

In initial studies, biofilms of BAA-42 MRSA were treated with 1000×MICconcentrations (1000 μg/ml) for each of daptomycin, linezolid, andvancomycin and 1×MIC (32 μg/ml) for PlySs2 lysin (without added reducingagent). All MIC values were determined using the broth microdilutionmethod described in the Clinical and Laboratory Standards Institute(CLSI) document M07-A9 (Methods for dilutional antimicrobial sensitivitytests for bacteria that grow aerobically. Volume 32. Wayne [PA]:Clinical and Laboratory Standards Institute [US], 2012). MRSA biofilmstreated for up to 4 hours are shown in FIG. 1 , up to 6 hours are shownin FIG. 2 , and up to 24 hours shown in FIG. 3 . The biofilm is clearedwithin 2 hours on treatment with PlySs2 lysin alone at 1×MIC 32 μg/ml(FIGS. 1, 2 and 3 ). No change in biofilm is evident visually in 4 hoursor 6 hours on treatment with 1000 ug/ml (1000×MIC) of daptomycin,vancomycin, or linezolid (FIGS. 1 and 2 ). This is consistent withprevious reports which have shown minimal sensitivity of biofilms tovancomycin at very high doses (10000 μg/ml) (Weigel L M et al (2007)Antimicrob Agents and Chemother 51(1):231-238).

Lower concentrations of PlySs2 lysin and daptomycin treatment wereevaluated against biofilms of MRSA strain BAA-42. Biofilms were treatedwith lower sub-MIC doses of PlySs2 for 0.5 hours, 1 hour, 4 hours and 24hours. As described above, BAA-42 biofilms were generated in 24 welldishes and the wells were treated with either PlySs2 lysin or daptomycinantibiotic (with proper media controls). For PlySs2, sub-MIC doses ofeither 3.2 ug/mL (a 1/10×MIC value) or 0.32 ug/mL (a 1/100×MIC value)were used. For daptomycin, either 1 ug/mL (a 1×MIC value) or 10 ug/mL (a10×MIC value) were used. The wells were incubated for up to 24 hours,washed, fixed and stained. The results are shown in FIG. 4 . Even at1/100^(th) the MIC of PlySs2 lysin, biofilm dissolution is observed.Significant dissolution is demonstrated with PlySs2 lysin 3.2 μg/ml (1/10×MIC) at 4 hours, and even some dissolution is observed with 0.32μg/ml ( 1/100×MIC) at 4 hours. With daptomycin concentrations up to10×MIC, no dissolution is seen.

Comparable MIC studies were completed using an alternativestaphylococcal lysin, particularly ClyS lysin, against ATCC BAA-42 MRSAbiofilms. The MIC of the ClyS lysin for this S. aureus strain is 32μg/ml. Polystyrene tissue culture plates were inoculated with 5×10⁵ CFUsof S. aureus strain ATCC BAA-42 per well (in Tryptic soy broth with 0.2%glucose) and incubated for 24 hours at 35° C. to allow biofilmformation. Resulting biofilms were washed 3 times to remove planktoniccells and treated with concentrations of ClyS lysin of 32 μg/ml, 3.2μg/ml, 0.32 μg/ml and 0.032 μg/ml (or media alone) for 24 hours at 35°C. Each well was washed and stained with 2% crystal violet. Crystalviolet stains the adherent biofilm material. The results using thevarious concentrations of ClyS are depicted in FIG. 14 . ClySeffectively disperses the biofilm at 32 μg/ml (1×MIC) and 3.2 μg/ml(0.1×MIC). Reduction in stained biofilm is also observed at 0.32 μg/mland somewhat at 0.032 μg/ml. The Staphylococcal lysin ClyS is capable ofdispersing and reducing S. aureus biofilm.

Example 2

Combinations of daptomycin plus lysin at sub MIC doses are evaluated onbiofilms. It has been found that PlySs2 lysin and daptomycin exert asynergistic lethal effect on planktonic S. aureus cells (U.S.Provisional Application Ser. Nos. 61/644,944 and 61/737,239). A seriesof experiments are undertaken to investigate whether this synergisticeffect can also target bacteria in a biofilm. The broth microdilutioncheckerboard method (Sopirala M M et al. (2010) Antimicob Agents andChemother 54(11):4678-4683) is applied to mature S. aureus biofilmsgrown in 96-well microtiter dishes. The activity of sub-MIC combinationsof lysin and daptomycin is examined against 18 hour biofilms of MRSAstrain ATCC BAA-42 grown in the manner described above with theexception that cells are grown in 0.2 ml suspensions. After biofilmformation, the wells are washed with 1×PBS and treated with PlySs2 anddaptomycin alone or in a series of combinations for 24 hours withoutaeration. The biofilms are then washed, fixed and stained as above toevaluate biofilm formation. The effect of sub-MIC drug combinations isthus evaluated by comparison to the effects of either drug alone atthose same sub-MIC concentrations.

Example 3 Mixed Biofilm Studies In Vitro

PlySs2 lysin is also used in combination with daptomycin to targetmulti-species biofilms. Biofilms often contain more than one bacterialspecies (Yang L et al (2011) FEMS Immunol and Med 62(3):339-347). PlySs2lysin and daptomycin are utilized to target biofilms comprised of thePlySs2- and daptomycin-sensitive S. aureus strain ATCC BAA-42 and thePlySs2-resistant, daptomycin-sensitive Enterococcus faecalis strain.While E. faecalis strains are sensitive to daptomycin in planktonicform, they are nonetheless resistant to daptomycin as a sessile memberof a biofilm. Only when the enterococci are released from a biofilm maythey become resistant to daptomycin. To test the ability of PlySs2 tomediate this release (and thus sensitize E. faecalis to daptomycin), thefollowing experiment is conducted.

Biofilms are generated as described above in 24 well dishes using aninitial inoculums of 1×10⁶ staphylococci and 1×10⁶ enterococci (eachalone and together). Biofilms are washed with PBS and treated withPlySs2 and daptomycin alone and in combination (using a series ofsub-MIC combinations) for 24 hours. After treatment, the biofilm wellsare separated into two fractions, including the non-adherent (includingboth living and dead bacteria) and the adherent (biofilm forms). Thenon-adherent fraction is plated for viability to determine relative CFUcounts for staphylococci and enterococci. The CFU counts generated arecompared to CFU counts for those biofilms treated with buffer controls.At the same time, the remaining biofilms are disrupted by sonication andplated for viability. In this manner, it can be determine if PlySs2mediates the release of E. faecalis from biofilms where it may be killedby the daptomycin.

Biofilms with lysin^(s)antibiotic^(s), lysin^(s)antibiotic^(R),lysin^(R)antibiotic^(s) combinations are also evaluated as noted below.

-   -   I. Staphylococcus/Enterococcus mixed biofilm—treatment with        lysin plus antibiotic as described above.    -   II. S. aureus/S. epidermidis mixed biofilm, or just S.        epidermidis biofilms are generated and evaluated. Experiments        are also performed as above using biofilms formed from S. aureus        and S. epidermidis bacteria.    -   III. Combination Staph+Strep bacteria biofilms, treatment with        PlySs2 and dapto or other antibiotics.        Experiments are performed as above using biofilms formed from        both S. aureus and S. pyogenes (or S. dysgalactiae). Since        both S. pyogenes (Group A streptococcus) and S. dysgalactiae        (Group B streptococcus) are sensitive to PlySs2, these        experiments will not utilize daptomycin. Rather, PlySs2 lysin is        evaluated alone to disrupt and kill organisms in a mixed biofilm        consisting of staphylococci and streptococci.

Example 4 In Vivo Catheter-Based Biofilm Models

Staphylococcus aureus infections associated with indwelling devices canbe very difficult to treat due to the recalcitrant nature of bacterialbiofilms to conventional antibiotics, and generally require removal ofinfected devices such as catheters. Courses of antibiotics can beadministered and may even appear to eliminate most of thedevice-associated bacteria, only to have a recurrence of infectionwithin a few days. This is believed to result from residual persisterstaphylococci in the biofilm outgrowing, repopulating the biofilm andreseeding the infection at the device site or elsewhere (Darouiche R O(2004) N Engl J Med 350:1422-1429). Therefore, a treatment that wouldrapidly kill staphylococci in biofilms and also be effective onplanktonic bacteria would be of great benefit. PlySs2 lysin isdemonstrated in the prior examples to rapidly and effectively clear S.aureus biofilms in vitro. This study assesses the ability of PlySs2lysin to eradicate established S. aureus biofilms on implanted cathetersin vivo in mice.

A catheter-based model was evaluated using catheters situatedsubcutaneous in flank, intraperitoneal or intramuscular into the thigh(modified from Zhu Y et al (2007) Infect Immunol 75(9):4219-4226). Thiscatheter-based murine model is used to assess the impact of PlySs2 onbiofilm viability in vivo. Prior to implantation, biofilms are grown invitro on segments of catheter tubing (PVC [polyvinyl chloride]containing DEHP [Di(2-ethylhexyl)phthalate] as a plasticizer; CareFusionSmartSite infusion set, #72023). The lumen of each 2 inch catheter isinoculated with 200 μl of Tryptic Soy Broth (TSB) supplemented with0.25% glucose containing 2×10⁷ CFU of S. aureus, and biofilms are grownfor 72 hours at 37° C. Alternatively, catheters are cut into 2 mmsegments and placed in 1.0 ml of inoculated TSB supplemented with 0.25%glucose, and catheter segments are passaged daily into fresh medium forthree days prior to implantation. Anesthesia is induced in 6-8 week oldBalb/c mice by intraperitoneal injection of 0.15 ml of 100 mg/kgketamine and 10 mg/kg xylazine (Butler-Schein). Catheter segments areimplanted subcutaneously in each flank of the mice, or alternativelyinto the intraperitoneal space or thigh muscle. Groups of 5-10 mice wereimplanted with biofilm. Mice are treated with an appropriate amount ofPlySs2, antibiotic or vehicle, or combination of PlySs2+antibiotic 1-24hours post implantation. All mice from each group were humanelysacrificed at 1-4 days post-infection. To quantify biofilm formation,infected catheters were removed immediately after sacrifice, gentlywashed three times in sterile PBS to remove non-adherent bacteria, andsubsequently placed in 5 ml of sterile PBS. Adherent bacteria areremoved from the catheters by sonication. The number of recoveredbacteria is then quantified by serial dilution and plate counting on theappropriate selective media. Alternatively, washed catheters werestained by 15 minute incubation in Methylene Blue, washed two times in 5ml of sterile PBS and visualized. Methylene Blue stain can then bequantified by destaining in 0.2 ml of 30% acetic acid at roomtemperature and the absorbance read at 600 nm. The extent of residualbiofilm mass is expressed as the absorbance reading at 600 nm divided bythe weight of the catheter segment (OD₆₀₀/gm).

FIG. 15 provides the results of such a catheter study wherein catheterswith S. aureus (MRSA strain ATCC BAA-42) biofilm grown for 3 days wereimplanted into subcutaneous space in mice and then treated at 24 hourspost implant. Mice were each implanted with 2 catheters and 2 miceevaluated for each of the following conditions: negative control (nobiofilm, no agent), PlySs2 control (no biofilm mock treated with PlySs2agent), vehicle only, PlySs2 administered intraperitoneally (IP), PlySs2administered intravenously (IV), and PlySs2 administered subcutaneously(SC). PlySs2 was administered as a single bolus of 100 μg (correspondingto 5 mg/kg in the mouse and ˜50 μg/ml dose). Catheters were removed 6hrs post treatment and stained with methylene blue. The relative amountof staining (visualized at 600 nm) under each condition is presented inFIG. 15 . Each of the IP, IV and SC doses reduced staining, with thesubcutaneous bolus resulting in elimination of staining in the catheterto near control levels.

Example 5

In another set of experiments, implanted jugular vein catheters in miceare pre-instilled with PlySs2 lysin to assess protection of mice frombiofilm infection with this pre-treatment. Using the jugular catheteranimal model described above, the catheters of jugular vein catheterizedmice are pre-treated with instillation of PlySs2 lysin in PBS 24 h priorto the S. aureus challenge. Control animals receive catheterspre-treated with PBS alone. On the day of the challenge, 2 h prior tothe challenge, all catheters are flushed with PBS to remove excessunbound lysin, and then the mice are challenged with S. aureus via thetail vein as described above. The challenged animals were sacrificed atvarious days after the bacterial challenge and the catheters and organsrecovered and bacteria quantified as described above.

Example 6

Staphylococcal endocarditis is a biofilm based infection that can beexperimentally induced in the aortic valve of rats (Entenza J M et al(2005) IAI 73:990-998). Briefly, sterile aortic vegetations are producedin rats and infusion pumps to deliver lysin are installed as described(Entenza et al). Endocarditis is induced 24 h later by i.v. challengewith 10⁵-10⁷ staphylococci. At either 24 or 48 hours after infection,lysin and/or antibiotics such as daptomycin, vancomycin, or linezolidare administered intravenously. Control rats receive buffer alone. Atvarious time points after infection up to 72 hours, animals aresacrificed and quantitative blood and vegetation cultures wereperformed. Bacterial densities are expressed as log₁₀ CFU per mL or gramof tissue, respectively.

Example 7

In order to compare relative biofilm eradication activities of PlySs2and standard-of-care antibiotics, a twenty-four hour time courseanalysis of PlySs2 and antibiotic activity was performed on MRSAbiofilms. Biofilms were generated in 24-well polystyrene plates byinoculating 10⁵ bacteria (MRSA strain ATCC BAA-42) into 0.5 mlTryptic-soy broth with 0.2% glucose (TSB+) per well and incubated for 24hours at 37° C. One plate was generated for each treatment time point tobe assessed (0, 0.5, 1, 2, 4, 6 and 24 hours). After 24 hours, media wasaspirated, wells were washed twice with 1×PBS, and the drug treatmentwas added and treatment time initiated. Indicated drug concentrations(1000×MIC for daptomycin, vancomycin or linezolid; 1×MIC for PlySs2lysin) in 1 ml MHB2 (or MHB2 supplemented to 50 ug CaCl₂) per ml) wereadded to each well and incubated for the indicated time periods beforeaspiration, 2 washes with 1×PBS, and air drying for 15 minutes. Wellswere stained with a 3% crystal violet solution in 1 ml for 5 min, thenaspirated, washed 3 times with 1×PBS, air dried for 15 minutes, andphotographed. All experiments were performed in duplicate. The resultsare shown in FIGS. 6 and 7 . Crystal violet staining of the wells isshown in FIG. 6 and quantitation of the dye retained in the wells of theplate is shown in FIG. 7 . PlySs2 at 1×MIC completely cleared thebiofilm by 2 hours while daptomycin, vancomycin, and linezolid at1000×MIC concentrations showed no biofilm clearance at 24 hours.

In order to determine the ability of sub-MIC concentrations of PlySs2 totreat biofilms, a twenty-four hour time course analysis was performed.Biofilms were generated in 24-well polystyrene plates by inoculating 10⁵bacteria (MRSA strain ATCC BAA-42) into 0.5 ml Tryptic-soy broth with0.2% glucose (TSB+) per well and incubated for 24 hours at 37° C. Oneplate was generated for each treatment time point to be assessed (30min, 1 hr, 4 hrs, 24 hrs). After 24 hours, media was aspirated, wellswere washed twice with 1×PBS, and PlySs2 was added and treatment timeinitiated. Indicated PlySs2 concentrations (0.1×MIC and 0.01×MIC) in 1ml MHB2, or media alone were added to each well and incubated for theindicated time periods before aspiration, 2 washes with 1×PBS, and airdrying for 15 minutes. Wells were stained with a 3% crystal violetsolution in 1 ml for 5 min and then aspirated, washed 3 times with1×PBS, air dried for 15 minutes, and photographed. All experiments wereperformed in duplicate. The results are shown in FIG. 8 . PlySs2 at0.1×MIC cleared the biofilm by 4 hours. PlySs2 at 0.01×MIC yieldedpartial clearance at 4 hours while full clearance was observed by the 24hour time point.

Example 8

The biofilm eradication activities were assessed for both PlySs2 anddaptomycin against MRSA biofilms grown on catheters. Biofilms weregenerated in 2 inch segments of catheter tubing (PVC [polyvinylchloride] containing DEHP [Di(2-ethylhexyl)phthalate] as a plasticizer;(CareFusion SmartSite infusion set, #72023) by inoculating 10⁵ bacteria(MRSA strain ATCC BAA-42) into 0.2 ml Tryptic-soy broth with 0.2%glucose (TSB+) per segment and incubated for 72 hours at 37° C. Allsamples were set up in duplicate for either staining with methylene blueor quantitation of CFUs. After 72 hours, media was flushed out, segmentswere washed with 1 ml of 1×PBS, and treatment was added. Indicated drugconcentrations (1×MIC and 1000×MIC for daptomycin, 1×MIC for PlySs2) in0.2 ml Lactated Ringer's solution were added to each segment andincubated for 24 hours before flushing, and washing with 1 ml 1×PBS.Duplicate samples were then examined as follows: To assess biofilmeradication, segments were stained with a 0.02% methylene blue solution(in water) in 0.22 ml for 15 min. Segments were then flushed, washed 3times with dH₂0, air dried for 15 minutes, and photographed. Toquantitate the amount of live cells retained within the residualbiofilms, duplicate segments were treated with 0.22 ml lysis buffer (100ug/ml lysostaphin in Lactated Ringer's Solution) for 8 minutes. Next,0.1 ml samples were removed, added to 96-well solid white polystyreneplate, and mixed with 0.1 ml of Promega BacTiter-GloLuciferin/Luciferase reagent and relative light units (RLUs) wereimmediately measured (as specified by the kit manufacturer'sinstructions) and compared to a previously generated standard curvecorrelating RLU values to known concentrations of bacteria. In thismanner, an estimation of bacterial CFUs in each biofilm was determined.

The results are shown in FIGS. 9A and 9B. Relative biofilm staining isshown in FIG. 9A. PlySs2 completely cleared the biofilm from thecatheter at 1×MIC, while daptomycin did not remove significant biofilmeven at 1000×MIC. As seen in FIG. 9B, PlySs2 at 1×MIC took the CFUs downto the 100 CFU/ml, which is the limit of detection, while no CFUreduction was seen with daptomycin at 1×MIC and a two log reduction from100 million to 1 million CFU/ml was observed at 1000×MIC daptomycin.

To determine lowest amount of PlySs2 needed to eradicate biofilm fromcatheters, a titration experiment was performed (FIG. 10 ). Biofilmswere generated in 2 cm segments of DEHP catheter tubing by inoculating10⁵ bacteria (MRSA strain ATCC BAA-42) into 0.2 ml Tryptic-soy brothwith 0.2% glucose (TSB+) per segment and incubated for 72 hours at 37°C. After 72 hours, media was flushed out, segments were washed with 1 mlof 1×PBS, and drug treatment was added. Indicated drug concentration(1×, 0.1×, 0.01×, 0.001×, 0.0001× and 0.00001×MIC amounts of PlySs2) in0.2 ml Lactated Ringer's solution were added to each segment andincubated for 24 hours before flushing, and washing with 1 ml 1×PBS.Segments were stained with a 0.02% methylene blue solution (in water) in0.22 ml for 15 min, before being flushed, washed 3 times with dH20, airdried for 15 minutes, and photographed. The amount of PlySs2 need tofully eradicate the biofilm as determined by staining was 0.01×MIC (0.32ug/ml) (FIG. 10 ). A similar titration analysis performed withdaptomycin (1×, 10×, 100×, 1000×, 5000×MIC daptomycin) showed thatconcentrations of daptomycin as high as 5000×MIC (5 mg/ml) did notremove the biofilm (FIG. 11 ).

For quantitation of CFUs remaining after biofilm treatment with lysin orantibiotic, duplicate segments as assessed in FIGS. 10 and 11 weretreated with 0.22 ml lysis buffer (100 ug/ml lysostaphin in LactatedRinger's Solution) for 8 minutes. Next, 0.1 ml samples were removed,added to 96-well solid white polystyrene plate, and mixed with 0.1 ml ofPromega BacTiter-Glo Luciferin/Luciferase reagent and relative lightunits (RLUs) were immediately measured (as specified by the kitmanufacturer's instructions) and compared to a previously generatedstandard curve correlating RLU value to known concentrations ofbacteria. In this manner, an estimation of bacterial CFUs in eachbiofilm was determined. The titration analysis confirmed the results ofmethylene blue staining and is provided in FIG. 13 . PlySs2 is active atremoving biofilm bacteria down to a 0.01×MIC concentration whiledaptomycin is completely ineffective up to concentrations of 5000×MIC.

A time course analysis of PlySs2 activity against MRSA catheter biofilmswas then performed (FIGS. 12A and 12B). Biofilms were generated in 2inch segments of DEHP catheter tubing by inoculating 10⁵ bacteria (MRSAstrain ATCC BAA-42) into 0.2 ml Tryptic-soy broth with 0.2% glucose(TSB+) per segment and incubated for 72 hours at 37° C. Two samples wereset up for each indicated time point (0 min, 5 min, 15 min, 30 min, 60min, 90 min, 2 hrs, 3 hrs, 4 hrs, 5 hrs) to accommodate methylene bluestaining and CFU quantitation. After 72 hours, media was flushed out,segments were washed with 1 ml of 1×PBS, and treatment was added. PlySs2(1×MIC concentration, or 32 ug/mL) in 0.2 ml Lactated Ringer's solutionwere added to each segment and incubated for indicated time pointsbefore flushing, and washing with 1 ml 1×PBS. Duplicate samples werethen examined at each time point as follows: segments were stained witha 0.02% methylene blue solution (in water) in 0.22 ml for 15 min.Segments were then flushed, washed 3 times with dH20, air dried for 15minutes, and photographed. Duplicate segments were treated with 0.22 mllysis buffer (100 ug/ml lysostaphin in Lactated Ringer's Solution) for 8minutes. Next, 0.1 ml samples were removed, added to 96-well solid whitepolystyrene plate, and mixed with 0.1 ml of Promega BacTiter-GloLuciferin/Luciferase reagent and relative light units (RLUs) wereimmediately measured (as specified by the kit manufacturer'sinstructions) and compared to a previously generated standard curvecorrelating RLU value to known concentrations of bacteria. In thismanner, an estimation of bacterial CFUs in each biofilm was determined.The time course analysis revealed a progressive removal of stainablebiofilm from the catheters at 1×MIC PlySs2 over time, with full removalby 60 minutes (S 12A). The CFU analysis revealed a similar progressivetime course, with CFU values at the limit of detection (100 CFU/ml) by60 minutes (FIG. 12B).

Example 9

To determine the stability of PlySs2 in a simulated catheter setting,PlySs2 was incubated at various concentrations in Lactated Ringer'ssolution at 37° C. After 7 days, the lytic activity of PlySs2 wasassessed by adding 1×10⁵ staphylococci, incubating for 4 hours, thentreating with proteinase K to remove residual PlySs2, and serialdilution and plating for viability. The resulting CFU value for eachcondition was divided by 1×10⁵ to determine the % Loss of Activity.

The results are tabulated below in TABLE 6. After a 7 day incubation inLactated Ringer's solution at 37° C., undetectable activity losses wereobserved for the 10× and 100×MIC concentrations of PlySs2, while a 58.3%loss was determined for the 1×MIC sample.

TABLE 6 PlySs2 Stability at 37° C. in Lactated Ringer's SolutionTREATMENT % LOSS OF ACTIVITY (7 days) 1X MIC 58.3 10X MIC <0.002 100XMIC <0.002

The above indicates that PlySs2 is active and stable at least up to 7days in a simulated catheter setting and can effectively killStaphylococci and thereby prevent bacterial colonization even after anextended period of time incubating in Lactated Ringer's, an exemplarystandard care IV and flush solution.

Example 10

A time course study was conducted to evaluate luminal sterilization in acatheter to assess the viability of bacteria that are dislodged from thebiofilm and are suspended in the liquid phase of the lumen after or uponlysin treatment. In FIGS. 12A and 12B described above, it wasdemonstrated that the biofilm (adherent to the walls) is lost and fullydispersed by 1 hour. In the present study, sterilization (complete kill)of bacteria in the lumen, as evaluated by CFU analysis which detectslive cells, occurs approximately between 6 and 24 hours. Biofilms wereformed with strain ATCC BAA-42 for 3 days at 37° C. Biofilms were washedwith 1×PBS (to remove planktonic cells) and treated with either lactatedringer's solution (buffer control) or lactated ringer's solutioncontaining PlySs2 lysin (at a 1×MIC concentration) or daptomycin (at aIX MIC concentration) and also with PlySs2 lysin (at a 10×MICconcentration). Biofilms were treated for up to 24 hours and CFUsevaluated at 2 minutes, 15 minutes, 30 minutes, 1 hour, 2 hrs, 6 hrs and24 hours. At each time point, the lumenal contents of the catheters wereremoved and plated for viability. FIG. 16 provides the results for IXMIC (32 μg/ml), IX MIC daptomycin, and 10×MIC (320 μg/ml) leveltreatments versus buffer alone.

Example 11

Lysin was evaluated for effectiveness against Staphylococcus epidermidisbiofilms. Biofilms of various S. epidermidis strains were generated inpolystyrene 24-well microtiter plates and treated with PlySS2 lysin todetermine the minimal inhibitory concentration (MIC) and biofilmeradicating concentration (BEC) of PlySs2 against each strain. Theresults are tabulated below in TABLE 7 against over twenty distinct S.epidermidis strains. The MIC (in micrograms/mil) was determined andcalculated using standard CLSI method for broth microdilution asdescribed and referenced in the Examples above. The biofilm eradicatingconcentration (BEC) of PlySs2 (in micrograms/ml) is the lowestconcentration of a dilution range that completely destroys a 24 hourbiofilm of the indicated strains.

To determine BEC, 24 h biofilms were grown in 24 well plates, washed 2×with PBS, and treated with or without PlySs2 (dilution range) preparedin Lactated Ringers Solution. The treated plates were incubated at 37°C. (ambient air) for 24 hours, washed with PBS and stained with CrystalViolet (CV) for 15 minutes. The CV stain was next solubilized with 1 mLof 33%

TABLE 7 CFS Type Designation MIC BEC 166 Staphylococcus epidermidisEnvironmental lab contaiminant; na 5.12 NY, NY, 16S rRNA sequencing 224Staphylococcus epidermidis HER 1292 512 5.12 225 Staphylococcusepidermidis HPH-6 128 0.512 226 Staphylococcus epidermidis HPH-5 5125.12 227 Staphylococcus epidermidis HCN-4 >512 5.12 272 Staphylococcusepidermidis NRS53 (VISE) 128 0.215 280 Staphylococcus epidermidis NRS101(MRSE) 128 0.512 300 Staphylococcus epidermidis NRS8, (VISE) 32 0.512313 Staphylococcus epidermidis NRS34 (VISE) 8 0.512 533 Staphylococcusepidermidis NRS6; (VISE); bloodstream USA >512 0.512 552 Staphylococcusepidermidis ATCC #12228 (MSSE) na 51.2 769 Staphylococcus epidermidisNRS101 64 0.512 1152 Staphylococcus epidermidis ATCC-14990 na 5.12 1154Staphylococcus epidermidis ATCC-49461 na 5.12 1161 Staphylococcusepidermidis NRS850-VCU028 na 5.12 1164 Staphylococcus epidermidisNRS853-VCU041 na 5.12 1165 Staphylococcus epidermidis NRS854-VCU045 na5.12 1168 Staphylococcus epidermidis NRS857-VCU065 na 0.512 1174Staphylococcus epidermidis NRS864-VCU112 na 51.2 1184 Staphylococcusepidermidis NRS874-VCU126 na 5.12 1185 Staphylococcus epidermidisNRS875-VCU127 na 5.12 1186 Staphylococcus epidermidis NRS876-VCU128 na0.512 MIC = minimum inhibitory concentration of PlySs2 (in μg/ml)calculated using standard CLSI method for broth microdilution. na,indicates the data is not available BEC = Biofilm eradicatingconcentration of PlySs2 (in μg/ml) is the lowest concentration of adilution range that completely destroys a 24 hour biofilm of theindicated strainsacetic acid in each well, and absorbance (OD_(600nm)) was read using 200uL of the solubilized CV. Percent biofilm was determined by dividing theabsorbance of the well with the absorbance of the no lysin well (biofilmcontrol). The BEC was determined as the value that showed >75% clearingof the biofilm.

These results demonstrate the potent activity of PlySs2 lysin against S.epidermidis biofilms; notably, the potent activity extends to strainswith high PlySs2 MIC epidermidis biofilms; notably, the potent activityextends to strains with high PlySs2 MIC levels. These data indicatePlySs2 will be active against a wide range of S. epidermidis biofilms.

S. epidermidis biofilms in catheters were treated with PlySs2 andevaluated using methods similarly as described above for the S. aureusstudies. S. epidermidis does not produce biofilms on catheters asrobustly as the S. aureus strains previously described, however biofilmgrowth did occur and could be evaluated.

The results of S. epidermidis (strain CFS 313 NRS34, which is avancomycin intermediate sensitive S. epidermidis (VISE) strain) biofilmstudies on catheters treated with PlySs2 at 10×MIC and below are shownin FIG. 17 . S. epidermidis biofilm is destroyed at PlySs2concentrations down to 0.1×MIC. The MIC here is 8 ug/ml. A similarresult and comparable level of activity was observed with S. aureusstrain CFS 218 (MRSA strain ATCC BAA-42.).

Example 12

The results of a biofilm prevention assay are presented in FIG. 18 . S.aureus MRSA strain BAA-42 (5×10⁵ bacteria/ml) was inoculated in 2 ml ofTSB+0.2% glucose into each well of a row of a 24 well plate. LysinPlySs2 was added immediately (at concentrations 1×MIC (32 ug/ml),0.1×MIC, 0.01×MIC, 0.001×MIC and 0.0001×MIC and then incubated for 6hours at 37° C. in ambient air. Wells were washed with PBS, stained withCrystal Violet, and photographed to evaluate biofilm development undereach of the conditions. Buffer control was also evaluatedsimultaneously. In this study, the bacteria and lysin PlySs2 (differentconcentrations) are added at the same time and biofilm formation isallowed to proceed for 6 hours. As demonstrated in FIG. 18 ,preincubation with 1× and 0.1×MTC PlySs2 can effectively and completelyprevent the subsequent formation of biofilm. Thus not only can PlySs2eradicate mature biofilms, it can prevent de novo biofilm formation aswell.

Example 1-3

In addition to biofilms generated by BAA-42 MRSA as described above,additional S. aureus strain biofilms were evaluated for susceptibilityto PlySs2 lysin. Each of MRSA strains CFS 553 (ATCC 43300) (FIG. 19 )and CFS 992 (JMI 5381) were evaluated in catheter studies using methodsas described above. In each instance, 3 day-old biofilms were washed andtreated with indicated PlySS2 concentrations for 4 hours. The IX MIC forstrain ATCC 4330 is 16 μg/ml and the IX MIC for strain JMI 5381 is 32μg/ml. As shown in FIGS. 19 and 20 , these alternative MRSA strainbiofilms were susceptible to PlySs2 and Plyss2 eradicated and fullydispersed the catheter biofilm at levels of 10×MIC, 1×MIC, and 0.1×MIC.The biofilms were significantly reduced in each strain using 0.01×MICPlySs2.

Example 14

Biofilms were generated on catheter tubing (PVC with DEHP asplasticizer) as above and evaluated for PlySs2 sensitivity by scanningelectron microscopy (SEM). The three-day-old biofilms of MRSA strain CFS218 (MRSA strain ATCC BAA-42) on the catheter surface were treated witha 1×MIC concentration (ie, 32 ug/ml) of PlySs2 in Lactated Ringer'sSolution for either 30 seconds or 15 minutes before the treatment waswashed away and the remaining biofilm was fixed with gluteraldehyde.After fixation on the catheter surface, samples were further processedand analyzed by scanning electron microscopy at 5000× magnification(FIGS. 21A-21C). Treatment with buffer alone (ie, Lactated Ringer'sSolution alone) is included as a control. As shown in FIGS. 21A-21C, thePlySs2 treatment rapidly diminishes the MRSA biofilm (within 30 seconds,FIG. 21B) and by 15 minutes FIG. 21C almost completely removes thebiofilm.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method for prevention, disruption or treatmentof a gram-positive bacterial biofilm comprising contacting a biofilmwith a composition comprising a lysin polypeptide capable of killingStaphylococci, wherein the biofilm is effectively prevented, dispersedor treated.
 2. The method of claim 1 wherein the lysin polypeptide isPlySs2.
 3. The method of claim 2 wherein the lysin polypeptide comprisesan amino acid sequence as set out in FIG. 5 (SEQ ID NO: 1) or variantsthereof having at least 80% identity to the polypeptide of FIG. 5 (SEQID NO: 1) and effective to kill the gram-positive bacteria in thebiofilm.
 4. The method of claim 1 wherein the composition furthercomprises one or more antibiotic.
 5. The method of claim 4 wherein theantibiotic is selected from daptomycin, vancomycin, and linezolid. 6.The method of claim 1 further comprising contacting the biofilm with oneor more antibiotic.
 7. A method of preventing or reducing gram-positivebacterial biofilm formation comprising contacting a medical device,catheter, or implant with a composition comprising a lysin polypeptidecapable of killing Staphylococci wherein the lysin is PlySs2.
 8. Themethod of claim 7 wherein the lysin polypeptide comprises an amino acidsequence as set out in FIG. 5 (SEQ ID NO: 1) or variants thereof havingat least 80% identity to the polypeptide of FIG. 5 (SEQ ID NO: 1) andeffective to prevent or reduce the formation of bacterial biofilm or theattachment and growth of bacteria on the medical device, catheter, orimplant.
 9. The method of claim 7 wherein the composition furthercomprises an antibiotic.
 10. The method of claim 9 wherein theantibiotic is selected from daptomycin, vancomycin, and linezolid or arelated compound.
 11. A composition for use in prevention, disruption ortreatment of a gram-positive bacterial biofilm comprising a lysinpolypeptide comprising an amino acid sequence as set out in FIG. 5 (SEQID NO: 1) or variants thereof having at least 80% identity to thepolypeptide of FIG. 5 (SEQ ID NO: 1) and effective to kill thegram-positive bacteria in the biofilm.
 12. The composition of claim 11further comprising one or more antibiotic.
 13. The composition of claim12 wherein the antibiotic is selected from daptomycin, vancomycin, andlinezolid or a related compound.
 14. A composition for prevention,disruption or treatment of a Streptococcal or Staphylococcal bacterialbiofilm comprising a lysin polypeptide comprising an amino acid sequenceas set out in FIG. 5 (SEQ ID NO: 1) or variants thereof having at least80% identity to the polypeptide of FIG. 5 (SEQ ID NO: 1) and effectiveto kill the Staphylococcal or Streptococcal bacteria.
 15. Thecomposition of claim 14 further comprising one or more antibiotic.